Preparation of compositions to treat and treatment of osteoarthritis using adipose-derived stromal vascular fraction cells

ABSTRACT

A method for preparing a treatment composition for treatment of osteoarthritis including un-cultured stromal vascular fraction cells from adipose tissue includes direct aspiration of material of a centrifugally-formed pellet phase including stromal vascular fraction cells from enzymatically-digested adipose tissue from an internal containment volume of a portable apparatus through an aspiration tube inserted into the pellet phase and into a fluid receptacle located outside of the internal containment volume. The treatment composition may be administered into or in the vicinity of a patient&#39;s joint to be treated for osteoarthritis.

CROSS REFERENCE TO RELATED APPLICATIONS

This patent application is a continuation of U.S. patent applicationSer. No. 14/403,861 entitled “Tissue Processing Apparatus And Method ForProcessing Adipose Tissue”, filed Nov. 25, 2014, which is a U.S.national stage of International Patent Application No. PCT/US2013/058292filed Sep. 5, 2013, which claims a benefit of U.S. Provisional PatentApplication No. 61/697,716 titled “Tissue Processing Apparatus AndMethod For Processing Adipose Tissue”, filed Sep. 6, 2012. Thisapplication incorporates by reference the entire contents, and everyportion thereof, of each and every U.S. patent application andinternational patent application identified herein. This applicationincorporates by reference the entire contents, and every portionthereof, of each and every one of the following patent applications:U.S. patent application Ser. No. 14/403,861; International PatentApplication No. PCT/US2013/058292; U.S. Provisional Patent ApplicationNo. 61/697,716; International Patent Application No. PCT/US2013/021156;U.S. patent application Ser. No. 14/370,694; U.S. Provisional PatentApplication No. 61/585,566; International Patent Application No.PCT/US2011/043451; U.S. patent application Ser. No. 13/808,550; and U.S.Provisional Patent Application No. 61/363,150.

FIELD OF THE INVENTION

The invention relates to methods of processing adipose tissue to preparean osteoarthritis treatment composition including stromal vascularfraction cells and methods for treating a patient for 37(osteoarthritis.

BACKGROUND OF THE INVENTION

Adipose tissue is recognized as a promising source of stem cells with atleast multi-potent differentiation potential. Lipoasperate obtainedduring a lipoplasty procedure, such as lipo surgery, may be processed toprepare a so-called stromal vascular fraction (SVF) that is rich inleuko stromal vascular cells, which include stem cells. Processing toprepare SVF may include washing lipoasperate with saline solution,followed by enzymatic digestion of washed tissue using collagenase, andcentrifuging digested material to prepare SVF in the form of acentrifuged pellet. Such collection and processing of tissue involvesseveral steps with transfer of contents between different processcontainers for different tissue collection and processing steps, whichis cumbersome and provides significant opportunities for error orcontamination.

Some attempts have been made to design portable containers in whichlipoaspirate may be collected and then processed within the container todigest tissue and prepare a concentrate rich in leuko stromal vascularcells. Potential benefits of using such portable containers include areduced need to transfer material between containers to performdifferent process steps and a reduction in the need for multiple,specially-designed processing containers. However, such multi-stepprocessing in portable containers faces significant equipment andprocess design and operating limitations, especially when attempting toprocess relatively large volumes of adipose tissue at one time. Desiredleuko stromal vascular cells, including stem cells, are sensitive toprocessing conditions and viability of recovered cells may suffersignificantly if processing is not adequately controlled. Also, recoveryof cells from the container is of critical importance. Significantpotential exists for loss of valuable cells to recovery from thecontainer, such as by cells adhering to internal equipment and surfaceswithin the container. One problem with multi-step processing in a singleportable container is that the container design and processingoperations must accommodate the different requirements of each of thedifferent process steps to be performed in the single container, andwith severe volume constraints in relation to a practical size for sucha portable container. In contrast, processing systems that involvetransfer of contents between multiple different containers forperformance of different process steps benefit from an ability tooptimize equipment and process design for each process container that isdedicated to performance of a single step of an overall process.Therefore, multi-container processing has significant advantages interms of step-by-step equipment and process optimization. Moreover, amulti-container design is better suited for automation, for example withautomated transfer of processed material through conduits betweendifferent process containers or with automated control of processparameters for uniformity and process control.

SUMMARY OF THE INVENTION

Disclosed are portable apparatus, uses of such apparatus and methods forprocessing of human biological material, and which biological materialmay contain stringy tissue, such as is the case with adipose tissue.Stringy tissue such as collagen adds complexity to processing, forexample due to potential plugging of filters and interference withseparation of desired cellular components. Processing may includeapplications to prepare a washed or cleaned biological material or torelease and prepare a concentrate of portions of a biological materialfeed. In the context of adipose tissue, processing may be directed topreparing washed tissue for a fat graft or to prepare a concentrateproduct rich in leuko stromal vascular cells. Leuko stromal vascularcells may be referred to herein also as stromal vascular cells orstromal vascular fraction cells.

Obtaining a high recovery in a concentrate of viable leuko stromalvascular cells from adipose tissue and effective removal of suchconcentrate material of such a concentrate from the container in anoperationally convenient manner have been significant challenges formulti-step processing in a single container. Also challenging has beenproviding a portable container apparatus design that providesversatility in being adaptable both for applications involvingpreparation of washed adipose tissue for fat grafts and for applicationsinvolving digestion of adipose tissue and release and concentration ofleuko stromal vascular cells. The presence of stringy tissue components,such as collagen, in adipose tissue complicates processing, andespecially in the context of separating leuko stromal vascular cells forrecovery in a concentrate at a high yield in a high quality concentrateproduct from a multi-step processing container. Also even afterpreparation of such a cell concentrate in a multi-step processingcontainer, removal of the cell concentrate material from the containeris complicated by the presence of other materials that may remain in thecontainer after preparation of the cell concentrate and possiblephysical loss of leuko stromal vascular cells through adherence of cellsto exposed surfaces within the container (e.g., surfaces of containerwalls, filters, mixers or other apparatus components disposed in thecontainer).

A first aspect of the disclosure is provided by an apparatus forprocessing human biological material containing stringy tissue. Thestringy tissue may comprise collagen and/or other stringy tissuecomponents, for example as is typically the case with lipoaspirate. Theapparatus includes a tissue collector disposed in a tissue retentionvolume of a container. The presence of stringy tissue presents asignificant problem in relation to recovery of leuko stromal vascularcells from lipoaspirate, especially when processing large tissue volumesthrough multiple processing steps in a single container. Such stringytissue may tend to collect on and clog a filter through which stromalvascular cells pass for collection. Problems with stringy tissue may bereduced to some degree by using a pre-filter upstream of the containerto filter out stringy tissue before introduction into the container.However, such pre-filters are not easy to use and introduce additionalcomplexity for the medical professional performing a lipoplastyoperation. Also, even with the use of such a pre-filter, some stringytissue may still be introduced into the container and may significantlyimpact cell collection in the container. The inclusion of a tissuecollector in the container according may significantly reduce or even insome cases eliminate the need and complexity of using a separatepre-filter to remove some or all of the stringy tissue prior tointroduction of tissue into the container for processing.

The apparatus of the first aspect includes a container having aninternal containment volume, the internal containment volume including atissue retention volume and a filtrate volume. A filter is disposedwithin the internal containment volume with the tissue retention volumeon one side of the filter and the filtrate volume on another side of thefilter with the tissue retention volume and the filtrate volume being influid communication through the filter. An inlet port in fluidcommunication with the tissue retention volume is configured to accessthe tissue retention volume for introducing human biological materialinto the tissue retention volume. A suction port in fluid communicationwith the filtrate volume is configured to access the filtrate volume forsuctioning material from the filtrate volume. A tissue collector isdisposed in the tissue retention volume and rotatable relative to thecontainer in at least a first direction of rotation about an axis ofrotation. The tissue collector may include at least one toothed memberthat sweeps through a portion of the tissue retention volume when thetissue collector is rotated in the first direction. The toothed membermay be configured with a plurality of teeth to collect and retainstringy tissue when the tissue collector is rotated in the firstdirection in contact with human biological material containing thestringy tissue disposed in the tissue retention volume.

A number of feature refinements and additional features are applicableto the apparatus of the first aspect. These feature refinements andadditional features may be used individually or in any combination. Assuch, each of the following features may be, but are not required to be,used with any other feature or combination of the first aspect or anyother aspect of the disclosure.

Each such toothed member may include at least 3, at least 4 or at least5 teeth and may include an open space between the teeth of each pair ofadjacent said teeth. Each such toothed member may include up to 10, upto 20 or up to 25 or more such teeth. A leading edge of a toothed membermay be made up with at least 10 percent, at least 20 percent, at least30 percent, at least 40 percent, at least 50 percent, at least 60percent or more of open spaces. Such a leading edge of a toothed membermay be made up of no more than 99 percent, 90 percent, 80 percent, 70percent, 60 percent or 50 percent of teeth. By space on a leading edgeof a toothed member made up of such open spaces, it is meant the spacebetween tops of the teeth, and likewise the space on a leading edge madeup of a toothed member refers to the space along the edge occupied bythe tops of the teeth.

The tissue collector may include at least 1, at least 2, at least 3 orat least 5 such toothed members. The tissue collector may include up to6, up to 10 or even more such toothed members. When the tissue collectorincludes multiple toothed members, some or all of such toothed membersmay have the same or a different configuration, for example in relationto member size, tooth design, number of teeth, teeth density, or otherdesign features.

The apparatus may include at least 1 or at least 2 or more of suchtissue collectors. The apparatus of the first aspect may include only 1,up to 2 or more such tissue collectors when the apparatus includes aplurality of tissue collectors two or more of the tissue collectors maybe of the same configuration or of different configurations.

A toothed member may have a first end located radially toward the axisof rotation and a second end located radially away from the axis. Such asecond end may be located a distance from the axis of at least 1centimeter, at least 2 centimeters, at least 3 centimeters or at least 5centimeters or more from the axis. Such second end may located adistance from the axis of up to 6 centimeters, up to 8 centimeters, upto 10 centimeters or more from the axis.

Teeth may project toward a leading side, or edge, of the toothed memberwhen the tissue collector is rotated in the first direction. Teeth mayproject in a plane of rotation of the toothed member when the tissuecollector is rotated in the first direction. Teeth may have a height ina range having a lower limit of 1 millimeter, 2 millimeters, 3millimeters or 5 millimeters and an upper limit of 20 millimeters, 15millimeters or 10 millimeters relative to a bottom of an adjacent openspace.

The apparatus of the first aspect may have one or more than one mixingimpeller in the tissue retention volume. A mixing impeller may beconfigured to direct axial flow from the mixing impeller in a directiontoward the tissue collector. Such a mixing impeller may include at leastone portion configured to scrape a portion of the filter when the mixingimpeller is operated. Each such portion of a mixing impeller configuredto scrape a portion of the filter may include a peripheral edge portionof an impeller blade. At least a part of each such portion of the filtermay be in a tapered portion of the filter that is disposed in a taperedportion of the internal containment volume. The tissue collector andsuch a mixing impeller may be coaxial and rotatable about a common axisof rotation. A spacing along such an axis between such a mixing impellerand a toothed member of the tissue collector may be at least 0.25centimeter, at least 0.5 centimeter, at least 1 centimeter or at least 2centimeters. A spacing along the axis between such a mixing impeller anda toothed member of the tissue collector may be up to 3 centimeters, upto 5 centimeters or even more. Such a mixing impeller may extend to afirst radial distance from the axis and the tissue collector may extendto a second radial distance from the axis, with the second radialdistance being larger than the first radial distance. Such a secondradial distance may be at least 1 millimeter, at least 2 millimeters orat least 3 millimeters larger than such a first radial distance. Such asecond radial distance may be no more than 3 centimeters, no more than 2centimeters or no more than 1 centimeter larger than such a first radialdistance.

In addition to such a mixing impeller, which may be a first mixingimpeller, the apparatus of the first aspect may include one or moreadditional mixing impellers disposed in the tissue retention volume. Theapparatus of the first aspect may include a second mixing impellerconfigured to direct axial flow in a direction away from the tissuecollector when the rotatable shaft is rotated in the first direction.The tissue collector and such a second mixing impeller may be coaxialand rotatable about a common axis in the first direction. A spacingalong an axis between such a second mixing impeller and a toothed memberof the tissue collector may be at least 0.25 centimeter, at least 0.5centimeter, at least 1 centimeter or at least 2 centimeters. A spacingalong an axis between such a second mixing impeller and a toothed memberof the tissue collector may be up to 3 centimeters, up to 5 centimetersor even more. Such a second mixing impeller may extend to a third radialdistance from an axis that is at least 1 millimeter, at least 2millimeters or at least 3 millimeters smaller than a radial distancefrom the axis to which the tissue collector may extend. Such a thirdradial distance may be no more than 3 centimeters, no more than 2centimeters or no more than 1 centimeter larger than a radial distanceto which the tissue collector may extend.

The apparatus of the first aspect may be orientable in a firstorientation in which the inlet port and the outlet port are configuredfor access therethrough from above the container into the internalcontainment volume. The apparatus of the first aspect may be configuredto have all access to the internal containment volume be from above thecontainer in the first orientation.

The apparatus of the first aspect may include an extraction portconfigured for accessing the internal containment volume to removeprocessed biological material from the internal containment volume. Suchan extraction port may be configured for access therethrough from abovethe container into the internal containment volume when the apparatus isoriented in a first orientation. Access through such an extraction portmay be through a lumen extending through a rotatable shaft aligned withthe axis. In an alternative configuration, no access may be providedinto the internal containment volume through a lumen extending through arotatable shaft.

A permanent obstruction may be placed in such a lumen to prevent suchaccess. The apparatus may be configured for use to prepare a fattransfer, or fat graft, when such a permanent obstruction is placed insuch lumen through a rotatable shaft. Such a permanent obstruction maybe in the form of solder or a weld or a permanent plug. As analternative, a solid mixer shaft may be used instead of an obstructionin a lumen. Processed tissue in the tissue retention volume, for examplewashed adipose tissue for use in a fat graft, may be removed from thetissue retention volume from an auxiliary, or additional access, portprovided into the tissue retention volume. Such an additional accessport may provide access through a top of the container into the tissueretention volume. To remove processed tissue from the tissue retentivevolume adipose tissue, a syringe may be inserted into or mated with theadditional access port and the apparatus tipped to cause processedtissue in the tissue retention volume to collect in the vicinity of theadditional access port to be withdrawn into the syringe. As analternative processed tissue in the tissue retention volume may beremoved through the inlet port, which may be in a similar manner asdescribed for such an additional access port.

The filter may have a separation size of at least 70 microns, at least100 microns, at least 150 microns, at least 175 microns or at least 200microns. The filter may have a separation size of no larger than 800microns, no larger than 700 microns, no larger than 600 microns, nolarger than 500 microns, no larger than 475 microns, no larger than 450microns, no larger than 425 microns, no larger than 400 microns, nolarger than 350 microns, no larger than 300 microns or no larger than250 microns. For some applications, the filter may have a separationsize that is larger than 400 microns, for example for cell processingapplications when the apparatus of the first aspect is to be used to arecover a stromal vascular fraction concentrate from adipose tissue.Even though stromal vascular cells will easily pass through a 200 micronfilter, the larger filter size may be advantageous to promote recoveryof most or substantially all of the stromal vascular cells in thefiltrate volume. Smaller size filters may plug to a degree thatsignificantly reduces cell yield in terms of cell collection in andrecovery from the filtrate volume. In some other applications, thefilter may have a separation size of 400 microns or less, for examplefor processing adipose tissue for use in a fat graft, or fat transfer,operation. The filter may not be as susceptible to clogging in thoseapplications and a smaller filter size permits retention of desiredadipose tissue in the tissue retention volume. By separation size, it ismeant the size at which the filter effects separation between particlespassing through and particles rejected by the filter during normaloperation. The separation size may be determined by the size of openingsprovided in a surface filter, such as the mesh size of a mesh bag filteror of a rigid mesh screen filter.

The apparatus of the first aspect may be configured to be received in acentrifuge for centrifuging the container.

The apparatus of the first aspect may comprise human biological tissuecomprising stringy tissue disposed in the tissue retention volume incontact with the toothed member. The stringy tissue may comprisecollagen. Tissue to be processed in the apparatus of the first aspectmay comprise adipose removed from a patient during a lipoplastyprocedure (e.g., lipoaspirate). For example, the term tissue may be usedherein to refer to in-tact tissue, disrupted tissue, tissue fragmentsand biological fluids associated with or separate from tissue. Theapparatus may be orientable in a collection orientation for collectionof human biological material, or tissue, such as may comprise adiposetissue collected during a lipoplasty procedure. The collectionorientation is also referred to herein as an access orientation or afirst orientation, and the terms are used interchangeably. Forconvenience of description except as noted, the apparatus is describedas oriented in such a collection orientation. As such, relationalreferences such as to top, bottom, up, down, above, below, elevations,vertical, horizontal and the like are in relation to the apparatus asoriented in the collection orientation. The apparatus may be configuredsuch that the apparatus may be stably supported in the collectionorientation. For example, the apparatus may have a base configured forinterfacing with a flat, substantially horizontal surface (e.g., countertop or table top) to stably support the apparatus in the collectionorientation, or may be held in a mounting structure that maintains theapparatus in the collection orientation. Although such an orientation isreferred to as a “collection” orientation it should be appreciated thatuse of the apparatus is not limited to being oriented only in thecollection orientation or that only human biological material collectionmay be performed while the apparatus is oriented in the collectionorientation. The apparatus may be advantageously configured to permitperformance of many different operations with the apparatus when theapparatus is oriented in the collection orientation.

The apparatus of the first aspect may be used in a variety of processingapplications. The apparatus may, for example, be used for preparation ofconcentrated or separated portions of the collected human biologicalmaterial, for example to produce a stromal vascular fraction rich inleuko stromal vascular cells, including stem cells, derived from adiposetissue. As another example, the apparatus may be used for preparation ofa fat graft comprising adipose. The apparatus has a design thataccommodates retention of a target material (e.g., leuko stromalvascular cells or adipose) in a single container from collection throughpreparation of a desired product containing the target material. Bytarget material, it is meant some component or components from or someportion or portions of collected human biological material of interestfor recovery following processing in the apparatus, such as recovery ina concentrated or modified form relative to the collected humanbiological material (e.g., stromal vascular fraction concentrate rich instem cells and other leuko stromal vascular cells, cleanedadipose-containing fraction for fat grafting applications).

The apparatus of the first aspect may be used during multiple processingsteps to prepare, for example, a stromal vascular fraction concentrate(e.g., concentrate rich in leuko stromal vascular cells) from humanbiological material comprising adipose or a fat graft containingadipose, without the need to transfer a target material being processedbetween different containers for different processing steps. Theapparatus may be used initially to collect the human biological material(e.g. tissue and fluids) during a lipoplasty procedure or other tissueextraction procedure, or tissue that has already been extracted inanother procedure may be introduced into the apparatus for processing.The apparatus, and therefore also the container of the apparatus, may beportable and easily transportable between locations where collection ordifferent processing operations may be conducted.

The apparatus of the first aspect may have a collection volume withinthe filtrate volume (i.e., is a part of the filtrate volume). Thecollection volume may have a bottom elevation corresponding to a bottomelevation of the filtrate volume. The collection volume may have a topelevation that is lower than a bottom elevation of the tissue retentionvolume.

The apparatus of the first aspect may include an extraction port influid communication with the internal containment volume and configuredfor removing processed biological material from the internal containmentvolume. Any or all of the inlet port, the suction port and theextraction port may be configured for access therethrough from above thecontainer into the internal containment volume. The extraction port maybe located above a portion of the filter, so that the advancing tip of ahypodermic needle pierces the filter when the tip of the hypodermicneedle is advanced from the extraction port into the collection volume.The collection volume may include a nadir and the extraction port may bepositioned above the nadir so that the tip of a hypodermic needleinserted through the extraction port may be advanced vertically downwardto the vicinity of the nadir of the collection volume.

The apparatus of the first aspect may be configured for advancing ahypodermic needle through a lumen and out of the distal end of the lumento access the collection volume with an advancing tip of the hypodermicneedle. The distal end of the lumen may be located in the tissueretention volume above a portion of the filter, so that the advancingtip of the hypodermic needle may pierce and pass through the filter whenthe tip of the hypodermic needle exits the distal end of the lumen andis advanced from the distal end of the lumen into the collection volume.The collection volume may include a nadir, and an axis of the lumen maybe aligned so that the tip of a hypodermic needle exiting the distal endof the lumen may be advanced to the vicinity of the nadir of thecollection volume. The hypodermic needle may thus access the collectionvolume to permit injection of material into and/or aspiration ofmaterial from the collection volume (e.g., aspiration of stromalvascular fraction concentrate or other processed biological materialcollecting in the collection volume during processing).

The apparatus of the first aspect may be designed for single-use, andpiercing the filter with a hypodermic needle may beneficially provide asafety mechanism for preventing reuse, and risks associated therewith,by damaging the filter in a way that renders the filter unsatisfactoryfor reuse.

As noted, the suction port is in fluid communication with the filtratevolume. By the suction port being in fluid communication with thefiltrate volume, it is meant that the suction port is fluidly connecteddirectly to the filtrate volume, and not indirectly through the tissueretention volume and the filter. The fluid communication may be providedby a dedicated conduit extending from the suction port to a desiredlocation within the filtrate volume where it is desired to apply suctiondirectly to the filtrate volume. The suction port may be in fluidcommunication with a tapered portion of the internal containment volumethrough a conduit providing fluid communication from the suction port toa location within the filtrate volume that is also within the taperedportion of the internal containment volume. The conduit may extendthrough the filtrate volume from adjacent the suction port to such alocation within the filtrate volume. The suction port may be locatedabove the tapered portion of the internal containment volume. Thesuction port may be configured for access through the suction port fromabove the container. The suction port may be configured for connectionto a vacuum system to suction material from the filtrate volume, such asmaterial that passes through the filter from the tissue retention volumeto the filtrate volume.

The apparatus of the first aspect may include multiple suction ports.For example, the apparatus may include a first suction port as describedin the preceding paragraph that is in fluid communication with a firstlocation in the filtrate volume within the tapered portion of theinternal containment volume through a first conduit, and the apparatusmay include a second suction port through which components passingthrough the filter from the tissue retention volume to the filtratevolume may be suctioned from the filtrate volume through a secondconduit extending from the second suction port to a second locationwithin the filtrate volume. The second conduit may be configured topermit adjustment of the elevation of the second location within thefiltrate volume.

Any one or more of the inlet port, the suction port of other portsproviding access to the internal containment volume may be configuredfor access through the port from above. In this way, access through eachsuch port may be conveniently from above the apparatus, providing asignificant advantage to a user of the apparatus in that such a user mayfocus all access manipulations from above the apparatus while theapparatus is in a normal position in the collective orientation, forexample with the apparatus freestanding on a flat work surface such as atable or counter. Although such access from above the container may beat some angle relative to vertical, in a preferred implementation theaccess through such port is in a vertical direction from above thecontainer. In one preferred implementation, all access to the internalcontainment volume may be through access ports wherein each such accessport (e.g., inlet port, suction port, extraction port, other ports) isconfigured for access through the access port only from above thecontainer. In another preferred implementation, all access ports may beconfigured for access through each such access port in a verticaldirection from above the container.

The internal containment volume of the container may have a taperedportion that tapers in a downward direction. The tapered portion mayhave a cross-sectional area that tapers, or reduces in size, in adirection toward the bottom of a collection volume. The tapered portionof the internal containment volume may help to direct and concentratetarget dense material (e.g., dense cells, stromal vascular fraction)toward and into the collection volume. At least a portion of thecollection volume may be located within or below such a tapered portion.At least a part of the tapered portion may be located above thecollection volume. The tapered portion of the internal containmentvolume may have a conical shape or any other shape with across-sectional area that tapers to reduce in size in a direction towardthe bottom of the collection volume. In various implementations, atleast a part of the tapered portion may be located above the collectionvolume. The tapered portion may have a uniform taper geometry (e.g.,constant rate of taper) or may have a varying taper geometry (e.g.,varying rate of taper in the direction of the taper).

In some implementations, the internal containment volume may have atleast a first tapered portion and a second tapered portion that islocated vertically lower than the first tapered portion, wherein thefirst tapered portion has a greater rate of taper than the secondtapered portion. The first tapered portion may be defined at least inpart by a first internal wall surface of the container that is at afirst angle relative to horizontal when the apparatus is in an accessorientation in a range having a lower limit of 20°, 25°, 30°, 35°, 40°,or 45° and an upper limit of 65°, 60°, 55°, or 50° and the secondtapered portion may be defined at least in part by a second internalwall surface of the container that is at a second angle relative tohorizontal when the apparatus is in an access orientation in a rangehaving a lower limit of 50°, 60°, 65° or 70° and an upper limit of 89°,88°, 85° or 82°, provided that the second angle is larger than the firstangle. Such a first tapered portion, for example as viewed in a verticalplane cross-section, may be defined at least in part by opposing ones ofsuch first internal wall surfaces. Such a second tapered portion in sucha vertical cross-section may be defined at least in part by opposingones of such second internal wall surfaces. The second tapered portionmay be disposed partially or entirely within the filtrate volume. Thesecond tapered portion may include at least a portion of a collectionvolume within the filtrate volume or may be entirely within such acollection volume. The second tapered portion may be or may be a part ofa pellet well located in a bottom portion of such a collection volume.The volume within the second tapered portion of the internal containmentvolume may be in a range having a lower limit of from 0.2 percent, 0.3percent, 0.5 percent, 0.7 percent or 0.8 percent of the portion ofavailable processing volume of the container that is within the tissueretention volume and an upper limit of 2.5 percent, 2 percent, 1.5percent, 1.2 percent or 1.1 percent of the portion of such availableprocessing volume of the container that is within the tissue retentionvolume. Such a portion of the available processing volume within thetissue retention volume may be a volume capacity of the apparatus forhuman biological material feed (e.g., adipose tissue feed) that may beprocessed in the apparatus. For some implementations, the second taperedportion of the internal containment volume may have a volume in a rangehaving a lower limit of 0.3 cubic centimeter, 0.5 cubic centimeter, 0.7cubic centimeter, 0.8 cubic centimeter, 0.9 cubic centimeter or 1.0cubic centimeter and an upper limit of 5 cubic centimeters, 3 cubiccentimeters, 2 cubic centimeters, 1.5 cubic centimeters, or 1.3 cubiccentimeters. The second tapered portion may have a vertical dimensionwhen the apparatus is in an access orientation of at least 1 centimeter,at least 1.5 centimeters, at least 2 centimeters or at least 2.5centimeters. The second tapered portion may have a vertical heightdimension when the apparatus is in an access orientation of up to 10centimeters, up to 5 centimeters, up to 4 centimeters or up to 3centimeters. The internal containment volume may include a third taperedportion that is located below the second tapered portion that has agreater rate of taper than the second tapered portion. A third taperedportion may be defined at least in part by a third internal wall surfaceof the container that is at an angle relative to horizontal that issmaller than the second angle. The third angle may have a value asdescribed previously for the first angle, provided that the second angleis larger than the third angle. The third tapered portion may occupy thelowermost portion of a collection volume in the filtrate volume, whichmay be a lowermost portion in a pellet well. The third tapered portionmay have a vertical height dimension when the apparatus is in an accessorientation that is smaller than a vertical height dimension of thesecond tapered portion. The third tapered portion may have such avertical height dimension that is not larger than 1 centimeter or nolarger than 0.5 centimeter. The third tapered portion may have a volumethat is smaller than the volume of the second tapered portion. The thirdtapered portion may have a volume that is no larger than 0.5 cubiccentimeter, no larger than 0.3 cubic centimeter or no larger than 0.2cubic centimeter. The first tapered portion may have a vertical heightdimension below a bottom of the filter that is smaller than a verticalheight dimension of the second tapered portion, and such a verticalheight dimension of the first tapered portion may be at least 0.5centimeter or at least 1 centimeter. The first tapered portion maybeneficially help stromal vascular fraction materials to move into thesecond tapered portion when the apparatus is centrifuged. The secondtapered portion, and also the third tapered portion if present, may beor be part of a pellet well, as discussed below.

Surprisingly, it has been found that the material of a pellet phasecontaining a concentrate of leuko stromal vascular cells from adiposetissue, such as may be formed during centrifuging, may be directlyaspirated from a collection volume at the bottom of the filtrate volumewithout first removing overlying less-dense material phases and withoutdispersing the material of the pellet phase in a suspension liquid.Although the pellet phase may typically have a very high viscosity, ithas been found that it is possible to aspirate the pellet phasematerial, for example though a hypodermic needle, without first dilutingthe pellet phase material to reduce viscosity, and without detrimentalbreakthrough of overlying, low viscosity aqueous liquid phase during theaspiration. This permits significant simplification in processing toremove such pellet phase material in some implementations.

The internal containment volume of the apparatus of the first aspect mayinclude a pellet well that may help facilitate effective removal ofpellet phase material by direct aspiration. The pellet well may bedisposed in a bottom portion of the filtrate volume below a bottomelevation of the filter and accessible only from above when theapparatus is in an access orientation. Such a pellet well that may beconfigured as a relatively deep, narrow chamber to help facilitateeffective direct aspiration of pellet phase material, such as aconcentrate of leuko stromal vascular cells.

A pellet well may include a second tapered portion, and also optionallya third tapered portion, of the internal containment volume below afirst tapered portion, as described above.

The filtrate volume may include a lower tapered portion below a bottomelevation of the filter and above a top elevation of a pellet well. Thelower tapered portion of the filtrate volume may be defined by internalwall surfaces of the container that are each inclined relative tohorizontal at a maximum angle of no larger than 60° when the containeris in an access orientation. The lower tapered portion of the filtratevolume may be or include that portion of a first tapered portion of theinternal containment volume, as discussed above, that is located belowthe filter. At least a portion of the pellet well may be defined by awall surface of the container inclined relative to horizontal at anangle that is larger than the maximum angle when the apparatus is in theaccess orientation. The wall surface of the container defining at leasta portion of the pellet well may be inclined relative to horizontal atan angle of at least 70°, at least 75°, at least 80°, or at least 85°.The wall surface of the container defining at least a portion of thepellet well may be inclined relative to horizontal at an angle of 90°(vertical) or less than 90°, when the apparatus is in the accessorientation.

A pellet well may have a volume in a range having a lower limit of 0.3cubic centimeter, 0.5 cubic centimeter, 0.7 cubic centimeter, 0.8 cubiccentimeter, 0.9 cubic centimeter or 1.0 cubic centimeter and an upperlimit of 5 cubic centimeters, 3 cubic centimeters, 2 cubic centimeters,1.5 cubic centimeters, or 1.3 cubic centimeters.

A pellet well may have a vertical height dimension when the apparatus isin an access orientation of at least 1 centimeter, at least 1.5centimeters, at least 2 centimeters or at least 2.5 centimeters. Apellet well may have a vertical height dimension when the apparatus isin an access orientation of up to 10 centimeters, up to 5 centimeters,up to 4 centimeters or up to 3 centimeters.

A pellet well may have at least one portion with a vertical length of 1centimeter, a maximum horizontal dimension along the vertical length ofno larger than 5 millimeters and a minimum horizontal dimension alongthe vertical length of no smaller than 1.5 millimeters. Having at leastone such a portion may facilitate receiving a distal end of a hypodermicneedle or other aspiration tube in a relatively deep, narrow volume foraspiration of pellet phase material without significant prematurebreakthrough of less-dense aqueous liquid phase that may be disposedabove the pellet phase following centrifuging.

A tapered portion of the internal containment volume may have a taperedportion nadir corresponding with a bottom elevation of the internalcontainment volume. The bottom elevation of a collection volume maycorrespond with the bottom elevation of the internal containment volume.Wall surfaces of the container defining a tapered portion of theinternal containment volume may coverage at a point at the taperedportion nadir. This is a particularly beneficial configuration,especially for applications when target material is to be collected inand removed from the collection volume in the vicinity of the taperedportion nadir. Such a tapered portion nadir may be located in a pelletwell located at the bottom of a collection volume.

The apparatus of the first aspect may be configured with a veryconvenient size from a number of perspectives, and with efficient use ofthe internal containment volume to facilitate efficient collection ofbiological material and versatility in post-collection processing. Theapparatus may be sized for convenient hand transportation, such asbetween a location where human biological material may be collected toother, different locations, where various processing of collectedmaterial may be carried out. The apparatus may also be sized forconvenient manipulation by a person.

For many applications, the apparatus of the first aspect may be sizedand configured such that the internal containment volume has a volume ina range with a lower limit of 100 cubic centimeters, 200 cubiccentimeters, 250 cubic centimeters, 300 cubic centimeters, 500 cubiccentimeters, 600 cubic centimeters or 700 cubic centimeters and an upperlimit of 1500 cubic centimeters, 1300 cubic centimeters, 1100 cubiccentimeters, 1000 cubic centimeters, 900 cubic centimeters, 800 cubiccentimeters, 500 cubic centimeters, 400 cubic centimeters or 300 cubiccentimeters, provided that the upper limit is larger than the lowerlimit. One preferred range for many applications is for the internalcontainment volume to be in a range of 700 cubic centimeters to 1000cubic centimeters. Another preferred range for some applications is forthe internal containment volume to be within a range of from 100 cubiccentimeters to 400 cubic centimeters, such as for example to prepare aconcentrate of leuko stromal vascular cells for administration to thevicinity of a joint for treatment of osteoarthritis By internalcontainment volume, it is meant the total internal volume containedwithin the walls defining the container, including volume that isoccupied by internal hardware, such as for example may be occupied by amixing device, barrier member, suction conduits, barrier skirt, etc. Aswill be appreciated, less than all of the internal containment volumewill be available for processing within the internal containment volume.

The terms “available processing volume” or “useful volume” are usedinterchangeably herein to refer to the portion of the internalcontainment volume that is effectively available to receive and processhuman biological material and additives (e.g. wash other additives)during use of the apparatus of the first aspect for collection ofbiological material or for post-collection processing. This availableprocessing volume is equal to the internal containment volume lessportions of the internal containment volume occupied by hardware (e.g.,mixing device, filter, skirt, suction tubes, barrier member, etc.) andless unoccupied portions of the internal containment volume noteffectively accessible for occupation by biological material duringcollection operations or by biological material or additives duringpost-collection processing. For example, the available processing volumemay exclude a small volume at the top of the container that is above abottom extension of the inlet port into the internal containment volume.This small void space may be beneficial to permit space for fluid toslosh within the container when contents of the container may beinternally mixed or externally agitated (e.g., by a shaker table). Formany applications, the available processing volume may be in a rangehaving a lower limit of 75 cubic centimeters, 100 cubic centimeters, 200cubic centimeters, 300 cubic centimeters, 400 cubic centimeters, 500cubic centimeters, 600 cubic centimeters, 650 cubic centimeters or 700cubic centimeters and an upper limit of 1300 cubic centimeters, 1100cubic centimeters, 1000 cubic centimeters, 900 cubic centimeters, 850cubic centimeters, 800 cubic centimeters, 750 cubic centimeters, 600cubic centimeters, 500 cubic centimeters, 400 cubic centimeters or 300cubic centimeters, provided that the upper limit is larger than thelower limit. In one preferred implementation for many applications, theavailable processing volume may be in a range of from 700 cubiccentimeters to 850 cubic centimeters.

Advantageously, the apparatus of the first aspect may be configured sothat a large portion of the available processing volume is within thetissue retention volume, while still permitting a high level ofperformance for various processing operations. The tissue retentionvolume may comprise at least 60 percent, at least 65 percent or at least70 percent of the available processing volume with the container. Often,the tissue retention volume will comprise not more than 95 percent, notmore than 90 percent or not more than 85 percent of the availableprocessing volume. For many preferred implementations, the tissueretention volume may comprise a portion of the available processingvolume that is at least 50 cubic centimeters, at least 100 cubiccentimeters, at least 200 cubic centimeters, at least 300 cubiccentimeters, at least 400 cubic centimeters, at least 500 cubiccentimeters, at least 600 centimeters or at least 650 cubic centimeters.The apparatus may advantageously be configured with only a small portionof the available processing volume occupied by a collection volume,located below the filter. For example, the collection volume maycomprise no more than 10 percent, no more than 7 percent or no more than5 percent of the available processing volume.

For many preferred implementations the apparatus may have a collectionvolume that is no larger than 75 cubic centimeters, no larger than 50cubic centimeters, no larger than 30 cubic centimeters, no larger than20 cubic centimeters, no larger than 10 cubic centimeters or no largerthan 5 cubic centimeters. The collection volume may be at least 1 cubiccentimeter, at least 2 cubic centimeters, or at least 4 cubiccentimeters. In one preferred implementation, the collection volume maybe in a range of from 10 cubic centimeters to 30 cubic centimeters. Forother implementations, the collection volume may be smaller than 10cubic centimeters. Typically, the entire collection volume will make uppart of the available processing volume.

One significant area of medical application for use of the apparatus ofthe first aspect is to prepare leuko stromal vascular cell concentratefor use in the treatment of osteoarthritis, for example in the vicinityof a patient's joint. In some applications for treatment ofosteoarthritis, the apparatus may be configured with a relatively smallinternal containment volume designed to process a volume of adiposetissue to prepare a volume of leuko stromal vascular cells that may beappropriate for use in a single injection formulation for treatment ofosteoarthritis at a joint. In some implementations, the apparatus mayhave an internal containment volume with a volume in a range having alower limit of 150 cubic centimeters, 200 cubic centimeters or 250 cubiccentimeters and an upper limit of 400 cubic centimeters, 350 cubiccentimeters or 300 cubic centimeters. The apparatus may be designed witha tissue retention volume that includes a portion of the availableprocessing volume of the apparatus in a range having a lower limit of 50cubic centimeters, 75 cubic centimeters or 100 cubic centimeters and anupper limit of 250 cubic centimeters, 200 cubic centimeters 150 cubiccentimeters or 125 cubic centimeters. The apparatus may be designed tocollect a pellet phase volume, which may correspond with a pellet wellvolume, in a range of from 0.5 cubic centimeter, 0.75 cubic centimeteror 1 cubic centimeter and an upper limit of 2.5 cubic centimeters, 2cubic centimeters 1.5 cubic centimeters or 1.3 cubic centimeters.

The apparatus of the first aspect may be packaged within a hermeticenclosure, for example as packaged for transportation and storage priorto use. The apparatus may be sterilized prior to packaging andmaintained in a sterile environment within the hermetic enclosure atleast until the apparatus is removed from the hermetic enclosure foruse. The apparatus may be designed for a single use following removalfrom the hermetic enclosure. After such single use, the apparatus may bedisposed of in an appropriate manner.

A second aspect of the disclosure is provided by an apparatus forprocessing human biological material including a container having aninternal containment volume, the internal containment volume including atissue retention volume and a filtrate volume. A filter is disposedwithin the internal containment volume with the tissue retention volumeon one side of the filter and the filtrate volume on another side of thefilter with the tissue retention volume and the filtrate volume being influid communication through the filter. An inlet port in fluidcommunication with the tissue retention volume is configured to accessthe tissue retention volume for introducing human biological materialinto the tissue retention volume. A suction port in fluid communicationwith the filtrate volume is configured to access the filtrate volume forsuctioning material from the filtrate volume. The filtrate volumeincludes a pellet well in a collection volume located below a bottomelevation of the filter.

A number of feature refinements and additional features are applicableto the apparatus of the second aspect. These feature refinements andadditional features may be used individually or in any combination. Assuch, each of the following features may be, but are not required to be,used with any other feature or combination of the second aspect or anyother aspect of the disclosure.

The apparatus of the second aspect may be or include any feature or acombination of any features described with respect to the first aspect.The pellet well of the apparatus of the second aspect may be or have anyfeature or features described with respect to a pellet well for theapparatus of the first aspect. The apparatus of the second aspect mayhave a design that does not include a tissue collector as described withrespect to the first aspect.

A third aspect of the disclosure is provided by a method of processingadipose tissue to concentrate leuko stromal vascular cells associatedwith the adipose tissue. The method combines particular processing incombination with a portable container of an apparatus for processinghuman biological material containing stringy tissue, to addresssignificant design constraints associated with the use of portablecontainers for multi-step processing of adipose tissue. The method ofthe third aspect includes multi-step processing within a portablecontainer having a filter inside the container. The multi-stepprocessing includes washing the adipose tissue within the container toremove contaminants from the adipose tissue. After the washing, themethod includes digesting adipose tissue within the container,comprising adding to the container a volume of enzyme-containingdigestion medium to contact washed adipose tissue in the container.After permitting enzymatic digestion in the container for a retentiontime following adding the digestion medium, the method includesdisposing the container in a centrifuge and centrifuging the containerin the centrifuge to form density-separated phases within the container,the density-separated phases including lower-density material phases anda higher-density pellet phase comprising leuko stromal vascular cells.After the centrifuging, the method includes selectively removingmaterial of the pellet phase from the container.

The method particularly addresses processing within the constrainedcontext of multiple-step processing within a single portable container.The method may permit effective processing within such a portablecontainer in a manner to address inherent equipment and processingdesign problems associated with multi-step processing in portablecontainers and without excessive losses of cell viability or physicallosses of cells to adherence to equipment and container surfaces insidethe container.

A number of feature refinements and additional features are applicableto the third aspect of the invention. These feature refinements andadditional features may be used individually or in any combination. Assuch, each of the following features may be, but are not required to be,used with any other feature or combination of the third aspect or anyother aspect of the disclosure.

In preferred implementations, the portable container may be a containerof an apparatus of the first aspect or second aspect of the invention. Afirst side of the filter within the container may be a filtrate volumeand a second side of the filter in the container may be a tissueretention volume of a container of an apparatus of the first aspect orsecond aspect. Alternatively, the portable container may be other than acontainer of the apparatus according to the first aspect or secondaspect. The method may include, after centrifuging, removing thecontainer from the centrifuge prior to the selectively removing.

The selectively removing may include inserting an aspiration tube fromoutside of to inside of the container to contact the pellet phase insidethe container and aspirating at least a majority of material of thepellet phase through the aspiration tube to outside of the containerwithout suspending material of the pellet phase in a suspension liquid.Aspirating the material of the pellet phase without first suspending thematerial in a suspension liquid is sometimes referred to herein asdirect aspiration of the material of the pellet phase. Such directaspiration may be performed without removal of lower-density materialphases from above pellet phase, may be performed after removing some butnot all of the lower-density material phases or may be performed afterremoving all of the lower-density material phases. The lower-densitymaterial phases may include an aqueous phase above the pellet phase andprior to the aspirating, the lower-density aqueous phase may be notremoved from above the pellet phase. Such aqueous phase may be leftsubstantially in-tact within the container during the aspirating, andmay remain in the container following the aspirating. Some or all of thelower-density material phases may remain in the container after theaspirating. The inserting may comprise inserting the aspiration tubedownward into the container from above. When lower-density materialphases remain in the container during the aspirating, the aspirationtube may be inserted downwardly through the lower-density materialphases and into the pellet phase material located below thelower-density material phases. The aspiration tube may be a needle(e.g., hypodermic needle), cannula or other device with a fluidcommunication channel. For many applications, a 18 to 22 gaugehypodermic needle may be used for the aspiration tube. During theaspirating, the aspiration tube may be in fluid communication with afluid receptacle, and the aspirating may include collecting at least amajority of the material of the pellet phase in the fluid receptacle.Such a fluid receptacle may be a syringe or other fluid containmentapparatus. By selectively removing the material of the pellet phasewithout requiring prior removal of less-dense material phases above thepellet phase, the process operation of removing the pellet phasematerial may be considerably simplified and the potential for processingerrors and for loss of cells to adhesion to apparatus surfaces may besignificantly reduced. The fluid receptacle may be pre-loaded with adispersion medium that mixes with the material of the pellet phase inthe fluid receptacle when the dispersion material is introduced into thefluid receptacle during the aspirating. The dispersion medium may be aliquid medium to disperse and suspend the cells of the pellet phasematerial. The dispersion medium may be a gel or gel-like material inwhich the cells of the pellet phase material may disperse and beretained. The dispersion medium may be a delivery vehicle for thecellular material (e.g., leuko stromal vascular cells) of the pelletphase, and such cellular material may be administered to a patient in adelivery composition including the cellular material and the dispersionmedium. Some examples for a dispersion medium that may be preloaded intothe fluid receptacle include compositions that may be or include one ormore of the following, either alone or with other components: salinesolution (e.g., a balanced saline solution, Hank's Balanced Solution),crystalloid solution (e.g., Lactated Ringer's solution), hyaluronic acidand hyaluronic acid-based materials. Such hyaluronic acid-basedmaterials may be substrate or carrier compositions based on hyaluronicacid. Any of these listed materials for possible use as or inclusion ina dispersion medium may also be part of a final delivery composition foradministration to a patient. The volume of dispersion medium pre-loadedinto the fluid receptacle may be any convenient volume for theapplication. In some preferred implementations, the dispersion mediummay be present in a sufficient volume to prevent clumping of material ofthe pellet phase in the fluid receptacle. The dispersion medium in thefluid receptacle may have a volume such that a volume ratio of thevolume of the dispersion medium to the volume of the pellet phasematerial introduced into the fluid receptacle during the aspirating isat least 1:1, at least 2:1, or at least 3:1. Often, such a volume ratiomay be up to 10:1, up to 7:1, up to 5:1; up to 3:1 or up to 2:1. In someimplementations, the dispersion medium in the fluid receptacle may havea volume of at least 1 milliliter, at least 2 milliliters or at least 3milliliters. In some implementations, the dispersion medium in the fluidreceptacle may have a volume of up to 10 milliliters, up to 7milliliters, up to 5 milliliters, up to 3 milliliters or up to 2milliliters. A mixture formed in the fluid receptacle during theaspirating may be further processed to prepare a delivery compositionincluding material of the pellet phase or the mixture may be in the formof a delivery composition that is ready as prepared in the fluidreceptacle for direct administration to a patient, such as by directinjection from the fluid receptacle into the patient followingcompletion of the aspirating. Further processing may includecentrifuging the mixture formed in the fluid receptacle to reform apellet phase, separation of the pellet phase material from other,typically less-dense material phases, that form during the centrifuging,followed by formulation of the pellet phase with other components toprepare a desired delivery composition, which could include any of thecomponents that could be used as the dispersion medium or any othercomponents suitable for a delivery composition. When the deliverycomposition is to be injected into a joint to treat osteoarthritis, thedelivery composition may in some preferred implementations have a totalvolume in a range having a lower limit of 0.5 milliliter, 1 milliliteror 2 milliliters and an upper limit of 5 milliliters, 4 milliliters or 3milliliters. The delivery composition may include a volume of the pelletphase material in a range having a lower limit of 0.25 milliliter, 0.5milliliter, 0.75 milliliter, or 1 milliliter and an upper limit of 2.5milliliters, 2 milliliters or 1.5 milliliters. When the pellet phasematerial includes a concentrate of leuko stromal vascular cells, thedelivery composition will include a mixture of the different cellspresent in the leuko stromal vascular fraction, without purification andwithout culturing. This is distinguishable from other treatmentcompositions that may be prepared using only certain types of cellsseparated from the leuko stromal vascular fraction mixture or usingcultured cells.

In some implementations, selective removal of material of the pelletphase may involve removing one or more, or all, of the lower-densitymaterial phases layers prior to removing material of the pellet phasefrom the container. When lower-density material phases are removed priorto removal of material of the pellet phase, removing the lower-densitymaterial phases may include removing such lower-density material phasesfrom the container in sequence of increasing density, which may includesuctioning the lower-density material phases from the container throughopen end of a suction conduit disposed in the container. Preferably,such an open end of a suction conduit may be disposed in the containernot directly above the pellet phase, to reduce the possibility thatsuction created in the container would structurally disrupt the pelletphase. In some preferred implementations, the pellet phase remains inplace and stationary, relative to the container, while the lower-densitymaterial phases are removed. In some implementations, removing thelower-density material phases may include tipping the container duringsuctioning of lower-density material phases to promote flow of at leasta final suction portion of the lower-density material phases within thecontainer laterally away from the pellet and toward the open end of thesuction conduit. The container may include a corner located lateral tothe pellet phase, and which may be located at an elevation of thecontainer that is higher than the bottom elevation of the pellet phase,or even higher than a top elevation of the pellet phase. The tipping maypromote flow of fluid of the lower-density material phases laterallytoward the corner for suctioning from the vicinity of the corner intothe open end of the suction conduit. In some preferred implementations,the lower-density material phases may be removed through a top of thecontainer.

The method may include one or more steps other than or in addition toany or any combination of the steps noted above. Any such other oradditional step may be performed between any of the steps noted above ormay be performed prior to or after any of the steps noted above.

When lower-density material phases are removed from the container priorto removal of material of the pellet phase, the method may include,after the removing of the lower-density material phases, introducingaqueous suspension liquid into the container and dispersing cells of thepellet phase in the suspension liquid, such as to form a dispersion ofthe cells in the suspension liquid. The suspension liquid may beintroduced at a volume in a range having a lower limit of 1, 2, 3 or 5milliliters and an upper limit of 25, 20, 15 or 12 milliliters. A volumeof suspension liquid of about 10 milliliters may be used for manyimplementations. A volume ratio of the suspension liquid to the volumeof the pellet phase may be in a range having a lower limit of 1:1, 2:1,3:1 or 5:1 and an upper limit of 25:1, 20:1, 15:1 or 12:1. A volumeratio of about 10:1 may be used in many implementations. After beingdispersed in a suspension liquid, the suspension liquid with dispersedcells may be removed from the container. Preferably at least most of thesuspension liquid is removed from the container and more preferablysubstantially all of the suspension liquid and substantially all of thecells from the pellet phase are removed from the container with thesuspension liquid. The suspension liquid and dispersed cells may beremoved through a top of the container, even though suspension liquidand dispersed cells may be removed from a location adjacent a bottom ofthe container. This suspension liquid and dispersed cells may be removedupward through a hollow member disposed downward into the container, forexample through a hollow needle or cannula. In some implementations,such a hollow member may pierce and extend across the filter. Such ahollow member may be an aspiration tube, as described above, and may bein fluid communication with a fluid receptacle, as described above.

After the selectively removing, the pellet phase material removed fromthe container may be further processed and/or mixed with othercomponents as desired, for example to prepare a desired deliverycomposition for administration to a patient. When the pellet phasematerial is first suspended in suspension liquid in the container beforeremoval, the further processing may include centrifuging the mixture,recovering the pellet phase material and formulating the pellet phasematerial to prepare a delivery composition. Such a delivery compositionmay be or have features as described above.

For the digesting, digestion medium may be added to the container at avolume ratio of the volume of digest medium to volume of adipose tissuewithin the container is in a range of from 0.6:1 to 2:1 and wherein thedigestion medium provides from 150 to 300 collagen digestion units (CDU)per milliliter of catalytic volume, and wherein the catalytic volume isthe total of the volume of digestion medium and the volume of adiposetissue within the container.

The digesting may comprise, after adding the volume of digestion medium,permitting enzymatic digestion within the container for a retention timein a range of from 20 minutes to 50 minutes while the container isdisposed in a temperature controlled environment with the temperaturecontrolled environment maintained within a temperature range of from 32°C. to 38° C. and with at least occasional agitation of contents withinthe container. The digestion medium may provide collagen digestion units(CDU) per milliliter of catalytic volume within a range that is narrowerthan the range listed above. Such a range may have a bottom limit of150, 175 or 200 CDU and an upper limit of 300, 275 or 250 CDU. In someimplementations, the digestion medium may provide about 225 CDU permillimeter of catalytic volume. In this regard, the catalytic volume isthe total volume of the digestion medium added to the container and thevolume of adipose tissue already disposed within the container when thedigestion medium was added. For example, if the volume of digestionmedium added to the container equals the volume of adipose tissuealready disposed within the container, then the digestion medium willneed to contain a concentration of collagenase enzyme that is twice aslarge as the desired concentration relative to the catalytic volume. Aswill be appreciated, the adipose tissue as collected will haveassociated contaminants, but in preferred applications with thoroughwashing, the adipose tissue should be cleaned of most contaminants sothat substantially all of the volume of material on a tissue retentionside of the filter in the container may be adipose tissue.

The volume ratio of digestion medium to adipose tissue may be in anarrower range than that described above. Such a volume ratio may have alower limit of 0.6:1, 0.75:1 or 0.9:1 and may have an upper limit of2:1, 1.75:1, 1.5:1 or 1.25:1. For various implementations, the volumeratio of digestion medium to washed adipose tissue may be about 1:1.

The retention time during the digesting may be within a narrower rangethan that described above. The retention time may be in a range having alower limit of 20 minutes, 25 minutes or 30 minutes and an upper limitof 50 minutes, 45 minutes or 40 minutes. For various implementations,the retention time may be about 35 minutes.

The digesting may include continuous agitation of the contents duringsome portion or substantially all of the retention time. The agitationmay include mixing, periodically or continuously, with a rotatable mixerdisposed within the container. The agitation may include periodic orcontinuous movement of the container to cause agitation of contentswithin the container. The agitation may include shaking the container,such as on a warmer-shaker. A temperature controlled environment may beprovided by a warmer-shaker.

Temperature control may be implemented at various points in theprocessing of the method. Digestion medium when added to the containermay be within a temperature range having a lower limit 32° C., 33° C.,34° C. or 35° C. and an upper limit of 38° C. or 37° C. The temperaturewithin a temperature controlled environment during digestion may bemaintained in a narrower range than that stated above. A temperaturecontrolled environment may be maintained within a temperature rangehaving a lower limit of 32° C., 33° C., 34° C. or 35° C. and an upperlimit of 38° C. or 37° C. Wash liquid, when added to the container, maybe within a temperature range having a lower limit of 32° C., 33° C.,34° C. or 35° C. and an upper limit of 38° C. or 37° C.

The washing may include at least one wash cycle or multiple wash cycles,with each wash cycle comprising: adding a volume of aqueous wash liquidto the container to contact the adipose tissue within the container;mixing the wash liquid and the adipose tissue in the container; andremoving at least a majority of the wash liquid with contaminants fromthe container on a first side (e.g., filtrate volume) of the filter andretaining at least most of the adipose tissue in the container disposedon a second side of the filter (e.g., tissue retention volume). For eachwash cycle, a volume ratio of wash liquid addition may be controlled.The volume ratio of wash liquid addition refers to a volume ratio of thevolume of wash liquid to a volume of adipose tissue within the containerto which the wash liquid is being added during the wash cycle. Thevolume ratio of wash liquid addition may be in a range having a lowerlimit of 0.5:1, 0.7:1 or 0.8:1 and an upper limit of 4:1, 3:1, 2:1 or1.5:1. For many implementations, the volume ratio of wash liquidaddition may be about 1:1. A cumulative volume ratio of wash liquidaddition may be at least 2:1, or at least 3:1. The cumulative volumeratio of wash liquid addition refers to a sum of the volume ratios forall of the wash cycles.

The washing may include more than two wash cycles. In someimplementations, the washing may comprise at least three wash cycles.For many implementations, three wash cycles may be sufficient, while forother implementations, two wash cycles may be sufficient.

Each wash cycle may comprise removing wash liquid (preferably at least amajority of the wash liquid and more preferably substantially all of thewash liquid) by suctioning from the filtrate volume of the container onthe first side of the filter. During such suctioning, the wash liquidmay be removed through a top of the container.

Mixing the wash liquid may include operating a rotatable mixer disposedin the container. The rotatable mixer may be manually operable, such asby a handle attached to a rotating shaft disposed through a top of thecontainer. The mixing may include manually (hand) manipulating such ahandle to manually rotate the mixer within the container. In variouspreferred implementations, such mixing may be performed followingaddition of the wash liquid, and preferably shortly following suchaddition, to thoroughly mix the wash liquid and the adipose tissue beingwashed. Such a rotatable mixer may also be used to mix the digestionmedium and the adipose tissue following addition of the digestion mediumto the container, and preferably shortly after such addition, tothoroughly mix the digestion medium and washed adipose tissue to bedigested.

The wash liquid used during the washing may but need not be of the samecomposition for each wash cycle. The wash liquid may include one or moreadditives. For example the wash liquid for one of more of the washcycles may include one or more than one of an anti-clotting agent, anantibiotic and an antifungal. In some preferred implementations, for atleast one wash cycle, the wash liquid includes at least one of ananti-clotting agent, an antibiotic or an antifungal. In otherimplementation, for at least one wash cycle, the wash liquid includes ananti-clotting agent, an antibiotic and an antifungal. One preferredexample for an anti-clotting agent is heparin.

The method may include, not later than 50 minutes following adding ofthe volume of digestion medium, adding a stopping reagent to thecontainer to stop enzymatic activity within the container. The adding ofa stopping reagent to the container may be performed within a narrowertime period than that described above. The digestion medium may be addedwithin a time period not later than 45 minutes following adding thevolume of digestion medium, not more than 40 minutes following addingthe volume of digestion medium or not more than 35 minutes followingadding of the volume of digestion medium. The stopping reagent maycomprise human albumin. The stopping medium may be added in an amountsufficient to substantially stop enzymatic activity within thecontainer. The stopping reagent may preferably be added before thecentrifuging of the container following the digesting.

The container may be conveniently transported between differentlocations for performance of different processing at the differentlocations, and preferably may be manually transported by being carriedby a person. For example, a temperature controlled environment may belocated at one location and the centrifuge may be located at a differentlocation, and the method may comprise after the retention time in atemperature controlled environment, transporting the container from thatlocation to the location of the centrifuge for performance of thecentrifuging. As another example, one or more wash cycles may occur atyet a different location, and the method may comprise transporting thecontainer from the location of a wash cycle to the location of atemperature controlled environment. By transporting the container fromone location to another it is meant that the container, along withcontents of the container, are physically moved from one location to theother location, whether or not there are intermediate stops along theway.

The method permits convenient and controlled processing of significantquantities of adipose tissue in a convenient manner. The volume ofadipose tissue (including contaminants), disposed in the tissueretention volume of the container on the second side of the filter atcommencement of the washing may be in a range having a lower limit of50, 100, 150, 200 or 250 cubic centimeters and an upper limit of 700,600, 500, 400, 300 or 200 cubic centimeters, provided that the upperlimit is larger than the lower limit.

It should be appreciated that when reference is made to “adipose tissue”or a volume thereof in relation to a method of the invention thereference may be to in-tact adipose tissue and any associatedcontaminants that are present with the in-tact tissue. Thesecontaminants come from the biological materials extracted from subjectsto obtain the adipose tissue. Contaminants that may be associated withthe adipose tissue include for example blood, free lipids, smallparticles and debris and other materials that may have been collectedwith the adipose tissue or result from degradation during tissuecollection or processing operations. The amounts of these contaminantswill generally be higher in unwashed adipose tissue at the commencementof washing operations and will generally be lower at the commandment ofdigesting operations, following the washing.

A fourth aspect of the disclosure is provided by a method of processingadipose tissue using an apparatus of the first aspect or the secondaspect. The method includes processing in the container of theapparatus, including washing adipose tissue within the container toremove contaminants from the adipose tissue. The washed adipose tissuemay be used, for example, for preparing a fat graft.

A number of feature refinements and additional features are applicableto the fourth aspect of the invention. These feature refinements andadditional features may be used individually or in any combination. Assuch, each of the following features may be, but are not required to be,used with any other feature or combination of the fourth aspect or anyother aspect of the disclosure.

The washing may include any feature or combination of any featuresdiscussed in relation to the third aspect of the invention. After thewashing, washed adipose tissue may then be removed from the apparatus,before or after mixing additives into the washed adipose tissue, forexample for use in a fat graft.

A fifth aspect of the disclosure is provided by uses of concentrate ofleuko stromal vascular cells from adipose tissue for treatment ofosteoarthritis.

A treatment composition for use to treat osteoarthritis may comprise aconcentrate of a mixture of leuko stromal vascular cells as recoveredfrom processing of adipose tissue, for example without purification of aspecific cell type or cell types from the stromal vascular fractionmixture and without culturing cells. The composition may be or includeany delivery composition or feature thereof described in relation to thethird aspect or any other aspect of the disclosure.

A method for treating osteoarthritis may include administration to apatient of such a treatment composition. The administration may involveinjection of the treatment composition into or in the vicinity of ajoint to be treated for osteoarthritis. The administration may be byinjection from a fluid receptacle (e.g., syringe) into which pelletphase material is directly aspirated and in which the treatmentcomposition was prepared to include dispersion medium pre-loaded intothe fluid receptacle, for example as described above.

A sixth aspect of the disclosure is provided by a method for recoveringa concentrate product comprising leuko stromal vascular cells fromadipose tissue. The method includes directly aspirating from aprocessing container pellet phase material prepared from processingadipose tissue in the container. The pellet phase material may be orhave any feature or combination of any features of described withrespect to any other aspect of the disclosure. The pellet phase materialmay have been formed in the container by any method described withrespect to any other aspect of the disclosure. The container may be anapparatus as described with respect to any other aspect of thedisclosure. These and other aspects of the invention will be furtherunderstood by reference to the drawings and the exemplary descriptionprovided below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows top, perspective, side and end views of an embodiment of atissue collection and processing apparatus.

FIG. 2 shows another perspective view of the same tissue collection andprocessing apparatus as FIG. 1.

FIG. 3 shows the same tissue collection and processing apparatus as FIG.2 with a shell removed.

FIG. 4 shows the same tissue collection and processing apparatus as FIG.3 with a filter removed.

FIG. 5 illustrates various regions within the tissue collection andprocessing apparatus of FIG. 2.

FIG. 6 illustrates a needle inserted into a tissue collection andprocessing apparatus.

FIGS. 7A and 7B illustrate a translatable conduit in a tissue collectionand processing apparatus.

FIGS. 8-11 illustrate various operations in a method of processingtissue within a tissue collection and processing apparatus.

FIG. 12 shows top, perspective, side and end views of another embodimentof a tissue collection and processing apparatus.

FIG. 13 shows an exploded view of the same tissue collection andprocessing apparatus as FIG. 12.

FIGS. 14 and 15 illustrate a portion of another embodiment of a tissuecollection and processing apparatus including a rotatable tissuecollector.

FIG. 16 illustrates an example tooth configurationfor a toothed memberfor a rotatable tissue collector.

FIG. 17 shows a sectional view illustrating some features of anotherembodiment of a tissue collection and processing apparatus.

FIG. 18 illustrates the same tissue collection and processing apparatusas FIG. 17 showing a needle inserted into a pellet well to aspiratepellet phase material.

FIG. 19 is a generalized process block diagram of an embodiment of amethod of processing adipose tissue.

FIG. 20 is a generalized process block diagram of another embodiment ofa method of processing adipose tissue.

FIG. 21 illustrates another example tooth configuration for a toothedmember for a rotatable tissue collector.

FIG. 22 illustrates another example tooth configuration for a toothedmember for a rotatable tissue collector.

FIG. 23 illustrates another example tooth configuration for a toothedmember for a rotatable tissue collector.

FIG. 24 illustrates another example tooth configuration for a toothedmember for a rotatable tissue collector.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The following description of embodiments may be exemplified by referenceto collecting and processing tissue comprising adipose, but theprinciples described apply also to collection and processing of othertissue.

Reference is made to FIGS. 1-11 concerning features of an apparatus 300useful for collection of tissue comprising adipose removed from apatient during a lipoplasty procedure and/or for post-collection,multi-step processing of collected adipose in a single container. InFIG. 1, the apparatus 300 is illustrated in a collection orientation.The collection orientation is the orientation in which the apparatus 300may be placed during the collection of adipose removed from a patientduring a lipoplasty procedure. The apparatus 300 may also be placed inthe collection orientation during stages of the post-collectionprocessing of collected tissue as described below. Accordingly,subsequent references herein to the orientation of the apparatus 300,such as top, bottom, lower and upper, will refer to such a collectionorientation. As illustrated, the apparatus 300 has an apparatus heightHA, an apparatus length L, and an apparatus depth (or width) D. Theapparatus 300 also includes a suction port 302 and an inlet port 304.The suction port 302 and inlet port 304 are disposed on the top of theapparatus 300 when the apparatus 300 is in the collection orientation asillustrated in FIG. 1. In FIG. 1, and in certain other subsequentfigures, the ports are illustrated as having caps 328 thereon. Such caps328 are used to cover the various ports and may be removed and replacedas necessary during use of the apparatus 300.

The apparatus 300 includes a shell 306 and a lid 308. The shell 306 is aunitary bowl-like member where the only access into the interior, orcavity of the shell 306 is through the opening at the top of the shell306. As illustrated in FIG. 1, this opening at the top of the shell 306may be covered by the lid 308. The lid 308 and shell 306 may be rigid.The lid 308 and shell 306 are each preferably made of a clear polymericmaterial, such as a clarified polypropylene polymer composition, whichprovides low cellular adhesion and reasonable clarity. The lid 308 andshell 306 may be fabricated by injection molding. The lid 308 may beattached to the shell 306 in any appropriate manner, including snapping,clamping and/or gluing onto the shell 306. Together, the shell 306 andlid 308 form a container 322 with an internal containment volume 330(see FIG. 5 and accompanying discussion below) within the apparatus 300.The internal containment volume 330 is the volume within the cavity ofthe shell 306 covered by the lid 308, and is the volume available fordisposing both hardware and material tissue to be processed in thecontainer. This container may have a container height Hc. The shell 306may include a set of integral base supports 310 that may support theapparatus 300 in the collection orientation when the apparatus is placedon a horizontal surface. The apparatus height HA is larger than thecontainer height Hc by the distance of projections above the top of thecontainer for the inlet port 304, suction port 302, caps 328 and otherupward projecting features described below. The shell 306 may beconveniently designed to efficiently fit within a centrifuge bucket. Theprojections above the container height Hc may be configured so as not tointerfere with operation of such a centrifuge. As seen in FIG. 1, theapparatus length L is equal to the container length and the apparatusdepth is equal to the container depth (or width). As will beappreciated, the corresponding height, length and depth dimensions ofthe internal containment volume 330 will equal the height, length anddepth dimensions of the container less the corresponding thicknesses ofwalls of the shell 306 and lid 308. As shown in FIG. 1, some featuresmay be integrally formed with the lid 308. For example as shown in FIG.1 the suction port 302 and the inlet port 304 are integrally formed as aunitary fabricated piece with the lid 308. It should be appreciated thatsuch features may be provided as separate pieces and then assembled,such as by gluing or other means. For structural integrity, fabricationas a unitary piece is generally preferred.

FIG. 2 shows another perspective view of the apparatus 300 with the caps328 to ports removed and with an installed handle 382. FIG. 3 shows theapparatus 300 in the same orientation as in FIG. 2 with the shell 306and handle 382 removed. With the shell 306 removed, a filter 312 can beseen that is disposed within the internal containment volume 330. Thefilter 312 may have a separation size in a range appropriate for theinternal application (e.g., fat graft or preparation of cellconcentrate). The filter is preferably made of a mesh material. Onepreferred mesh material is a nylon mesh. Also visible within theinternal containment volume 330 is a suction port conduit 314 extendingdownward from the suction port 302. Additionally, as illustrated in FIG.3, all components of the apparatus 300, except for the shell 306, areinterconnected to the lid 308. In this regard, the subassembly shown inFIG. 3 may be assembled as shown and inserted into the shell 306.

FIG. 4 shows another perspective view of the apparatus 300. FIG. 4 showsthe apparatus 300 in the same orientation as in FIG. 3 with both theshell 306 and the filter 312 removed. With the filter 312 removed, aflow barrier skirt 324 extending downward from the lid 308 into theinternal containment volume 330 is visible. In an example, the flowbarrier skirt may extend between 5 mm and 50 mm downward from the lid308. The flow barrier skirt 324 may serve as an attachment point for thefilter 308 such that the filter 312 may be fixed relative to the lid308. The flow barrier skirt 324 may also serve to prevent material fromentering a tissue retention volume 332 (described below) and immediatelymoving through the filter 312 into the filtrate volume 334. The tissueretention volume 332 is that portion of the internal containment volume330 contained within the filter 312 and barrier skirt 324 below the lid308. The filtrate volume 334 is that portion of the internal containmentvolume 330 disposed outside of the filter 312 and barrier skirt 324.With the flow barrier skirt 324 in place, and material entering theinlet port 304 must at least move to below the lowest level of the flowbarrier skirt 324 before it is able to pass through the filter 312 intothe filtrate volume 334. The flow barrier skirt 324 may be part of afilter subassembly that includes the flow barrier skirt 324 and thefilter 312. This subassembly is mounted to the lid 308 with four screws326.

The filter 312 is asymmetric with respect to the lid 308 and shell 306in that it is configured to provide clearance between its left side (asviewed in FIG. 3) and the shell 306 for the suction port 302 and suctionport conduit 314. A portion of the filter 312 may be disposed about(e.g., rest on or around) a portion of the suction port conduit 314.

With the filter 312 removed (FIG. 4), a mixing device 316 can be seen.The mixing device 316 includes a rotatable shaft 318 and a set of mixingmembers 320. The axis of rotation of the rotatable shaft 318 may bethrough a central axis of the rotatable shaft 318. The mixing members320 are in the form of paddles extending outward from the rotatableshaft 318. Accordingly, when the rotatable shaft 318 is rotated, themixing members 320 will be rotated through the materials within thetissue retention volume 332 to aid in mixing the materials within theinternal containment volume 330, and in particular within the tissueretention volume 332. The rotatable shaft 318 extends from outside ofthe internal containment volume 330 through the lid 308 to the inside ofthe internal containment volume 330. As the rotatable shaft 318 isrotatable relative to the lid 308, the mixing members 320 fixed to therotatable shaft 318 are also rotatable relative to the lid 308. Therotatable shaft 318 may be made from a metal composition, such asstainless steel (e.g., grade 303, 304, or 316). Alternatively, therotatable shaft 318 may be made from a high-strength polymer compositionsuch as an Ultem™ resin product.

The rotatable shaft 318 may include a handle interface 380 (FIG. 3) thatmay provide an interface for the handle 382 (FIG. 2) to beinterconnected to the portion of the rotatable shaft 318 outside of theinternal containment volume 330. The handle interface 380 of FIG. 3 isin the form of a pair of parallel surfaces disposed about the portion ofthe rotatable shaft 318 outside of the internal containment volume 330.The handle 382 has a mating pair of interior parallel surfacesconfigured such that when the handle 382 is placed over the handleinterface 380, turning the handle 382 will result in turning therotatable shaft 318 and the mixing device 316. Such an interface 380also allows for the handle 382 to be removed from and replaced on thehandle interface 380 as needed during use of the apparatus 300.

FIG. 5 is a side schematic view of the apparatus 300 showing the mixingdevice 316 and filter 312 within the shell 306. The internal containmentvolume 330 is the entire volume within the shell 306 and under the lid308. Together, the portions of the shell 306 and lid 308 that containthe internal containment volume 330 are a container 322 of the apparatus300. The filter 312 divides and separates the internal containmentvolume 330 of the container 322 into the tissue retention volume 332disposed inside the filter 312, and a filtrate volume 334 disposedwithin the shell 306 on the outside of the filter 312. The filtratevolume 334 is that portion of the internal containment volume 330 intowhich filtrate enters after passing through the filter 312 from thetissue retention volume 332.

Disposed within the internal containment volume 330 at the bottom of theshell 306, below a level 350 that is at or below the lowest extent ofthe filter 312 (and therefore also below the lowest extent of the tissueretention volume 332), is a collection volume 336, such that thecollection volume 336 occupies the lowermost portion of the filtratevolume 334 located below the lowest elevation of the tissue retentionvolume 332.

The shell 306 has a tapered wall portion 338 that defines a taperedportion 340 of the internal containment volume 330, such that thecross-sectional area of the tapered portion 340 of the internalcontainment volume 330 tapers with a reducing cross-sectional area in adirection toward bottom of the container 322. By tapering, it means thatthe cross-sectional area in a horizontal plane (assuming the apparatus300 is in the collection orientation) becomes smaller in the directionof the taper (e.g., a direction orthogonal to the horizontal plane). Thetapered portion 340 of the internal containment volume 330 occupies theportion of the internal containment volume 330 below a level 352 wherethe tapered wall portion 338 meets a straight wall portion 342 of theshell 306. The tapered wall portion 338 is shown as having a flat,uniform inclined wall surface. The incline angle of surfaces of thetapered wall portion need not be uniform from the top to the bottom ofthe tapered portion 340 of the internal containment volume 330, and mayvary from top to bottom with portions with different incline angles, andmay have a curved surface, provided that the cross-sectional area isreducing in the direction of the taper. Also, the tapered wall portion338 need not be uniform around the perimeter of the tapered portion 340of the internal containment volume 330. For example, in the embodimentin FIGS. 3-5, the tapered wall portion 338 has a steeper incline on theends than on the front or back of the apparatus 300.

The shell 306 may comprise an upper portion 344 generally above a level354 and having a first wall surface portion 348 defining a correspondingupper portion 368 of the internal containment volume 330. Substantiallyall of the first wall surface portion 348 may have an incline relativeto horizontal of at least 75°. For example, substantially all of thefirst wall surface portion 348 may be substantially vertical (90°incline relative to horizontal). The shell 306 may include a lowerportion 346 located below the upper portion 344 and having a second wallsurface portion 358 defining a corresponding lower portion 360 of theinternal containment volume 330. The lower portion 360 may include thetapered wall portion 338 defining the tapered portion 340 of theinternal containment volume 330. Substantially all of the tapered wallportion 338 may preferably have an incline relative to horizontal in arange of from 30° to 60°, although other angles or curved surfaces maybe used. The tapered portion 340 of the internal containment volume 330may occupy substantially the entire lower portion 360 of the internalcontainment volume 330. At least a first portion 362 of the filter 312may be disposed in the upper portion 368 of the internal containmentvolume 330 and a second portion 364 of the filter 312 may be disposed inthe lower portion 360 of the internal containment volume 330. Thetapered wall portion 338 may form a nadir 384 at its lowest elevation.The nadir 384 may also be a nadir of the collection volume 336, thefiltrate volume 334, the container 322, and the internal containmentvolume 330.

The internal containment volume 330 may include an available processingvolume or “useable” volume 366 which may be the portion of the internalcontainment volume 330 that is usable and/or may normally be occupied bymaterials within the container 322 during normal use. For example, theavailable processing volume 366 may be the portion of the internalcontainment volume 330 below a level 356 that coincides with the bottomextension of a port through the lid 308 and that is not occupied byportions (e.g., internal hardware) of the apparatus 300 within theinternal containment volume 330, such as the mixing device 316, barrierskirt 324, filter 312 and suction port conduit 314. The top of theavailable processing volume may be at the elevation of the bottomextension of the inlet port 304, which may define a maximum fill levelwithin the internal containment volume 330.

The inlet port 304 in fluid communication with the tissue retentionvolume 332 through the lid 308 is configured for introducing tissuecomprising adipose directly into the tissue retention volume 332 duringa lipoplasty procedure. However, use of the apparatus 300 is not solimited, and the tissue may be introduced into the apparatus usingtissue previously collected in another container and transferred to theapparatus 300. An additional access port 372 in fluid communicationthrough the lid 308 with the tissue retention volume 332 provides anadditional route into the tissue retention volume 332, for example forintroduction of additives.

The suction port 302 is in fluid communication through the lid 308 withthe filtrate volume 334 via suction port conduit 314 extending from thesuction port 302 to within the tapered portion 340 of the internalcontainment volume 330 in the vicinity of the top of the collectionchamber 336. The suction port 302 is configured for connection to avacuum system, for example through connection of a suction conduitthrough which suction may be applied by a vacuum system to suction fromthe filtrate volume 334 material passing through the filter 312 from thetissue retention volume 332 into the filtrate volume 334.

The rotatable shaft 318 may include a filter contact member 376 (FIG. 5)that is offset from an axis of rotation of the rotatable shaft 318. Alower end of the filter contact member 376 may contact a portion of thefilter 312 as illustrated in FIG. 5. As the rotatable shaft 318 isrotated, the filter contact member 376 may rotate in a circular pathabout the axis of rotation of the rotatable shaft 318 remaining incontact with and moving along a portion of the filter 312. This contactmay cause the filter 312 to deform and such deformation and/or thecontact between the filter contact member 376 and filter 312 may causematerials that may have adhered to the filter 312 in this region tobecome dislodged from the filter 312. Thus, the filter contact member376 may assist in keeping the filter from clogging and increasing theeffectiveness of the filter 312.

The rotatable shaft 318 may include a lumen 374 therethrough. The top ofthe lumen 374 is visible in FIG. 3. The lumen 374 may have a distal end386 (FIG. 4) within the tissue retention volume 332 and a proximal end388 (FIG. 3) outside of the internal containment volume 330 and thus mayallow access to the tissue retention volume 332 therethrough. The lumen374 may be disposed along the central axis of the rotatable shaft 318.The lumen 374 thus provides a conduit for accessing the internalcontainment volume 330. As further described below, the lumen 374 mayprovide access for removing processed material from the internalcontainment volume 330. In that respect, the opening through the lid 308through which the rotatable shaft 308 extends acts as an extraction portthrough which access is provided via the lumen 374 that passes throughsuch opening. The apparatus 300 may include a plug 378 (shown in FIG. 4and not shown in FIG. 3) that may be placed in the proximal end 388 oflumen 374 to seal the lumen 374.

As shown in FIG. 6, a hypodermic needle 392 may be inserted through thelumen 374 and may be advanced out of the distal end 386 of the lumen 374and to pierce through the filter 312 to directly access the collectionvolume 336 (the volume under the line 410 in FIG. 6). Thus, thehypodermic needle 392 may be used to inject material into, or remove(aspirate) material from the collection volume 336. Additionally, as theaxis of the lumen 374 is vertically oriented, access to the collectionvolume 336 using the hypodermic needle 392 is by downward verticalinsertion into the lumen 374 from above the container. Such verticalinsertion coupled with the ability of the apparatus 300 to be placed ona flat surface in the collection orientation, allows for user-friendlyaccess to the collection volume 336, and helps avoid complications thatcould compromise operations to collect valuable processed material fromthe collection volume 430.

The hypodermic needle 392 may be interconnected to a syringe 394. Theproximal end 388 of the lumen 374 may include a tapered receptacleadapted to mate with a tapered tip of the syringe 394. In this regard,as shown in FIG. 6, the depth of penetration by the hypodermic needle392 into the collection volume 336 when the tapered tip of the syringe394 is in contact with the tapered receptacle of the lumen 374 may becontrolled by controlling the length of the hypodermic needle 392extending from the syringe 394. Additionally, the proximal end 386 ofthe lumen 374 may include a feature, such as a notch, to retain ano-ring (not shown) such that when the syringe 394 is positioned againstthe proximal end 388 of the lumen 374, the o-ring forms a seal betweenthe proximal end 388 of the lumen 374 and the syringe 394 (i.e., a sealthrough the o-ring between a wall surface in the tapered receptacle andan exterior wall surface of the tip of the syringe inserted into thetapered receptacle).

Turning to FIGS. 7A and 7B, the second suction port 370 includes atranslatable member 396 that may be translated up and down relative tothe lid 308 to vary the depth (elevation within the filtrate volume 334)at which material from the filtrate volume 334 is drawn through thesecond suction port 370. Examples of the various depths (elevations) atwhich the translatable member 396 may be positioned are illustrated inFIGS. 7A, 8, 9, and 11 and are discussed below in relation to methods ofusing the apparatus 300. The fit between the translatable member 396 andthe opening through the lid 308 of the second suction port 370 is suchthat the translatable member 396 may be readily translated up and downto a desired level, while maintaining a tight enough fit to allow avacuum applied to the translatable member 396 to adequately drawmaterial out of the filtrate volume 334.

In general, the parts discussed with reference to the apparatus 300 maybe made from any appropriate biocompatible material. In particular, theshell 306 may be made from a biocompatible transparent polymer materialto allow inspection of the contents therein. Screws 326, 392 and therotatable shaft 318 may be made from metal, such as stainless steel.Other parts of the assembly 300 pictured in FIG. 1 may be made fromappropriate biocompatible polymers.

Various exemplary dimensions of one specific nonlimiting example of anapparatus 300 will now be described with reference to FIGS. 1 and 5. Inthis example, the apparatus 300 has apparatus height HA of about 157 mm,an apparatus length L of about 145 millimeters, and an apparatus depth Dof about 126 millimeters. The containment volume height Hc is about 124millimeters. The example has an available processing volume 366 of about760 milliliters and a collection volume of about 23 milliliters. Theportion of the tissue retention volume 332 that coincides with theavailable processing volume 366 is about 580 milliliters. As will beappreciated, a milliliter is equal in volume to a cubic centimeter, andthe volumes listed here in milliliters may be equivalently stated ascubic centimeters.

In a method for processing tissue from a lipoplasty procedure using theapparatus 300, the tissue may be subjected to multi-step processingwithin the internal containment volume 330 to prepare within theapparatus 300 a concentrated product comprising at least one targetcomponent, or at least one target material, from the tissue. Tissue tobe processed may be introduced into the tissue retention volume 332through the inlet port 304. The tissue may be pre-filtered if desiredprior to being introduced into the tissue retention volume 332. Themethod may comprise washing tissue in the internal containment volume330 with a wash liquid. Optionally, the washing may include centrifugingthe apparatus 300. After washing, the method may comprise digestingtissue within the internal containment volume 330. After the digestion,the method may include centrifuging the apparatus 300 to prepare in thecollection volume 336 a concentrate product comprising at least onetarget component. For example the concentrate product may comprise, ormay consist essentially of, stromal vascular fraction from adiposetissue, and a target component may be stem cells from adipose tissue.

During the washing, the wash liquid may be added to the internalcontainment volume 330 to contact tissue within the tissue retentionvolume 332 and with at least a portion, preferably a majority, and morepreferably most, of the wash liquid passing through the filter 312 intothe filtrate volume 334. The wash liquid may wash one or more componentsfrom the tissue while retaining washed tissue in the tissue retentionvolume 332. The washed tissue may be retained in the tissue retentionvolume 332 by the filter 312. Wash liquid passing into the filtratevolume 334 may be removed from the filtrate volume 334, along with anycomponent or components washed from the tissue. Optionally, after addingthe wash liquid, the apparatus 300 may be centrifuged to facilitate ahigh degree of separation of the wash liquid from the tissue retained inthe tissue retention volume 332. Next, the wash liquid may be removedfrom the filtrate volume 334 by suctioning through the suction port 302of the apparatus 300. The washing may include multiple wash stages.During the washing, the mixing device 316 may be rotated by rotating thehandle 382 to mix contents of the internal containment volume and assistthe washing process.

During the digestion, an enzyme, such as for example collagenase, may beadded to the internal containment volume 330 through the additionalaccess port 372 or through the inlet port 304. During the digesting, themixing device 316 may be rotated to assist in the digesting process.

After adding the enzyme, the digesting may comprise agitating contentsof the containment volume of the apparatus 300 for a time and at atemperature sufficient for the digestion to proceed to an extent tosignificantly release the target component, or material, in the desiredform capable of passing through the filter 312. The agitating mayinvolve any method to agitate contents of the internal containmentvolume 330, including for example one or both of: (a) shaking theapparatus 300 to agitate the contents within the apparatus 300 and (b)mixing the contents within the apparatus 300 by rotating the mixingdevice 316 using the handle 382.

Post-digestion centrifuging promotes separation of the target componentfrom the digested tissue and passage of the target component through thefilter 312 for collection in the collection volume 336. The targetcomponent may include leuko stromal vascular cells (e.g., stem cells)from adipose tissue. As illustrated in FIG. 8, multiple material phasesmay collect within the filtrate volume 334. The first (bottom) materialphase may be a small layer of red blood cells 428 located in the regionof the filtrate volume 334 below the line 408. This volume below theline 408 occupies a bottom portion of the collection volume 336. Thesecond material phase may be a stromal vascular fraction layer 430 fromadipose tissue and may be located in the region of the filtrate volume334 below the line 412 and above the line 408. As will be appreciated,the red blood cell layer 428 and the stromal vascular fraction layer 430may not be divided by a sharp line, and the blood cell layer 428 maygrade into the lower portion of the stromal vascular fraction layer 430.This volume below the line 412 and above the line 408 also occupies aportion of the collection volume 336. The stromal vascular fractionlayer 430, or the stromal vascular fraction layer 430 together with thered blood cell layer 428, may be in the form of a pellet, and may bereferred to as a pellet phase. A third material phase may be an aqueouslayer 432 that occupies the region of the filtrate volume 334 below theline 406 and above the line 412. A fourth material phase may be adisaggregated adipose layer 434 that occupies the region of the filtratevolume 334 below the line 414 and above the line 406. A fifth materialphase may be an oil layer 436 that occupies the region of the filtratevolume 334 below the line 416 and above the line 414. The separatedphase layers as shown are provided to illustrate relative positioningand are not intended to represent an actual scale of the relative sizesof the phases, except that the red blood cell layer 428 and stromalvascular fraction layer 430 are contained within the collection volume336 and the other layers extend above the collection volume. As will beappreciated, the material phases 428, 430, 432, 434 and 436 are in orderof decreasing density, with red blood cell layer 428 being the mostdense phase and with the aqueous layer 432, the disaggregated a disposelayer 434 and the oil layer 436 all being less dense than the stromalvascular fraction layer 430.

The translatable member 396 of the second suction port 370 may beemployed to first remove the oil layer 436, then to remove thedisaggregated adipose layer 434, and then to remove the aqueous layer432. As illustrated in FIG. 8, the translatable member 396 may bepositioned such that the end of the translatable member 396 is disposedwithin the oil layer 436. Suction applied to the translatable member 396will remove fluid. As fluid is removed, the translatable member may belowered to remove additional fluid down to a desired level, which may beremoval of all or most of layers 436, 434 and 432. For example, once theoil layer 436 has been removed, the translatable member 396 may belowered into the disaggregated adipose layer 434 and then the aqueouslayer 432 for sequential removal of these layers. FIG. 9 illustrates theaqueous layer 432 partially removed (after already removing the toplayers 436 and 434 such that the top of the aqueous layer 432 is at line418. As another example, the translatable member 396 may be initiallyinserted to the position shown in FIG. 9 and suction applied until aportion of the aqueous layer 432 is removed and also the disaggregatedadipose layer 434 and oil layer 436 are removed above line 418,resulting in the arrangement of FIG. 9.

Once fully inserted into the filtrate volume 334, the translatablemember 396 may not be operable to remove a portion of the aqueous layer432 while the apparatus is in the collection orientation. Accordingly, auser may gently tilt the apparatus 300 as illustrated in FIG. 10 tofurther remove the aqueous layer 432. As illustrated, the stromalvascular fraction 430 below the line 412 may form a pellet which mayretain its position as the apparatus 300 is tilted. This attribute ofthe pellet allows the apparatus 300 to be tilted such that the aqueouslayer 432 flows laterally toward the translatable member 396 disposedproximate to the interface between the tapered wall portion 338 of theshell 306 and the straight wall portion 342 of the shell 306 asillustrated by line 420 in FIG. 10. Such tilting can allow suction to beapplied to the aqueous layer 432 without the suction substantiallyaffecting the stromal vascular fraction 430, which remains in place andstationary relation to the container. Once the aqueous layer 432 hasbeen satisfactorily removed, the apparatus 300 may be returned to itscollection orientation, as shown in FIG. 11, for removal of the stromalvascular fraction 430 from the collection volume 336.

To remove material of the pellet phase after the less-dense materialphases have been removed from above the pellet phase, the hypodermicneedle 392 may be inserted into the collection volume 336 from thesyringe 394 as illustrated in FIG. 6 and a diluent fluid (e.g.,suspension liquid) may be injected into the collection volume 336 suchthat the diluent fluid, stromal vascular fraction 430 and the layer ofred blood cells 428 together occupy at least a portion of the collectionvolume, and are preferably limited to the collection volume. Afterinjection of the diluent fluid, a user may gently tap the apparatus 300against a hard surface to cause the diluent fluid to mix with thestromal vascular fraction and the layer of red blood cells. A secondhypodermic needle may then be inserted through the lumen 374 and thediluent/stromal vascular fraction/red blood cell mixture may be removedfrom the apparatus 300, to complete the selective removal of material ofthe pellet phase from the container 322 relative to other, less-densematerial phases.

An alternative technique to remove material of the pellet phase afterthe less-dense material phases have been removed from above the pelletphase, is to insert the hypodermic needle 392 such as shown in FIG. 6into the pellet phase. The pellet phase may then be directly aspiratedthrough the hypodermic needle 392 into the syringe 394, to completeselective removal of material of the pellet phase from the container 322relative to the other, less-dense material phases. In this way, thematerial of the pellet phase may be removed from the container 322without suspending material of the pellet phase in a suspension liquid.

As a further processing alternative, the material of the pellet phase(red blood cells layer 428 and stromal vascular fraction 430) may beremoved from the container 322 without first removing the less-densematerial phases (the aqueous layer 432, disaggregated adipose layer 434and oil layer 436), or at least without removing all of those less-densematerial phases. For example, a hypodermic needle (similar to FIG. 6)may be inserted through the lumen 374 with the distal tip of the needledisposed in the pellet phase and the material of the pellet phase maythen be directly aspirated through the hypodermic needle and into asyringe in fluid communication with the hypodermic needle. Thistechnique may permit selective removal of the material of the pelletphase from the container 322 without first removing the lessdense-material phases and without suspending the material of the pelletphase in a suspension liquid. After removal of the material of thepellet phase, the less-dense material phases may remain inside thecontainer 322. Removal by such a technique significantly simplifiesprocessing, because the processing associated with removing theless-dense material phases (including tapping the container) may beeliminated, reducing potential processing errors and potential loss oftarget cells to adhesion to container or equipment surfaces.

Referring now to FIGS. 12 and 13, a further embodiment is shown for atissue collection and processing apparatus. As shown in FIGS. 12 and 13,a tissue collection and processing apparatus 450 has a collectionorientation in a freestanding, upright position as supported by basesupports 452. The apparatus 450 includes a lid 454 covering a bowl-likeshell 456, which make up a container having an internal containmentvolume under the lid within the shell. The apparatus includes a firstsuction port 458, a second suction port 460, an inlet port 462 and anauxiliary access port 464, which may be generally as describes forsimilar features of the apparatus 300 described with respect to FIGS.1-11.

The first suction port 458 is connected with a suction conduit 466extending from the first suction port 458 to within a tapered portion ofan internal containment volume of the apparatus 450. The second suctionport 460 is adapted to receive a translatable suction conduit, similarto the translatable member 396 described with respect to FIG. 7. Theapparatus 450 includes a filter 468 suspended from the lid 454 and whichdivides the internal containment volume in the apparatus between atissue retention volume disposed inside the filter 468 and a filtratevolume disposed on the other side of the filter 468. The apparatus 450includes a rotatable mixer disposed within the filtrate volume thatincludes propellers 470 connected to a rotatable shaft 472, which may berotated to operate the rotatable mixer and cause the impellers 470 tomix and circulate fluid within the internal containment volume of theapparatus 450. The rotatable shaft 472 includes an internal lumen thatextends from a proximal end outside of the container of the apparatus toa distal end in the tissue retention volume, to permit access into theinternal containment volume in a manner similar to that discussedpreviously for the apparatus 300 shown in FIGS. 1-7. A removable plug474 may be disposed in a proximal end of the lumen for sealing the lumenwhen the lumen is not in use. The rotatable shaft includes a handleinterface which may be interfaced with a hand-manipulable handle 476(FIG. 13) to operate the rotatable mixer. The apparatus 450 includesattached caps 478 which may be used to cap the first suction portion458, second suction port 460 and inlet port 462 as needed, such as toseal the container for transportation between processing locations orduring agitation on a warmer-shaker during digestion operations. Theapparatus 450 is operable substantially in the same way as describedpreviously for the apparatus 300 shown in FIGS. 1-11. The apparatus 450includes volume gradation markings 480 that indicate the volumecontained within the tissue retention volume (within the filter 468) upto different elevations of the container 450 when in the accessorientation.

Reference is now made to FIGS. 12-15. FIGS. 14 and 15 show aconfiguration for an alternative rotatable assembly that may be disposedin the tissue retention volume of a tissue retention apparatus, and forconvenience of description and brevity will be discussed in the contextof an alternative embodiment of the apparatus 450 shown in FIGS. 12 and13. Reference numerals used in FIGS. 14 and 15 are the same as used inFIGS. 12 and 13 for like features. FIGS. 14 and 15 show a rotatabletissue collector 482, which may alternatively be referred to as a tissuecomb or more particularly as a stringy-tissue comb. As shown in FIGS. 14and 15, the lid 454 has the first suction port 458, second suction port460, inlet port 462 and auxiliary axis port 464 providing access throughthe lid 454 into the internal containment volume of the container of theapparatus 450. The mixing propellers 470 are shown mounted on therotatable shaft 472. For convenience of description, in FIGS. 14 and 15the lower propeller is designated 470 a and the upper propeller isdesignated 470 b. Also shown is a terminal end of a lumen 484 thatpasses through the rotatable shaft 472 to provide access from above thelid 454 into the internal containment volume of the container of theapparatus 450.

As shown in FIGS. 14 and 15, the tissue collector 482 is disposed on theshaft 472 intermediate between the propeller 470 a and propeller 470 b.The propellers 470 may alternatively be referred to as impellers ormixing impellers. Also shown in FIGS. 14 and 15 is a rotationaldirection 488 represented by an arrow and showing a rotational directionfor rotating the shaft 472 to rotate the propellers 470 and the tissuecollector 482 during one possible operation of the apparatus. Therotational direction 488 corresponds with the directional arrows on aplate shown in FIG. 13 that is visible at the top of the device to showdirection of rotation for operation of the apparatus 450.

The tissue collector 482 includes four tissue collection members 486,which may also be referred to as tissue collection blades. The tissuecollection members 486 each includes a plurality of teeth 490 and openspaces 492, configured with an open space 492 located between each pairof adjacent teeth 490. The tissue collection members 486 thus have atoothed configuration that facilitates engagement and collection ofstringy tissue, such as collagen, when the tissue collector 482 isrotated in the rotation direction 488, such as may be affected byrotating the rotatable shaft 472 using the handle 476. The open spaces492 may alternatively be referred to as slots or recesses, and the openspaces 492 provide locations for stringy tissue engaging with the tissuecollector 482 to be collected and retained. As stringy tissue collectsin the open spaces 492, the stringy tissue may also tend to wrap aroundthe rotatable shaft 472 to assist retention of the stringy tissue. Bycollecting and retaining the stringy tissue using the tissue collector482, plugging of the filter 468 may be significantly reduced becauseless of the stringy tissue is available to collect on and plug thefilter 468. The teeth 490 each have a top (maximum protrusion of a tooth490 relative to the bottom of an adjacent open space 492) that is thusdisposed toward a leading edge of the corresponding member 486 when thetissue collector 482 is rotated in the rotational direction 488. Thebottom of an open space 492 may be the most recessed portion of the openspace relative to the top of an adjacent tooth 490 as defined by thesurface geometry of the member 486. In the configuration shown in FIGS.14 and 15, each member 486 includes six teeth 490 and five open spaces492.

With continued reference to FIGS. 12-15, features of one or both of thepropellers 470 may be configured to assist collection of stringy tissueby the tissue collector 482 and to reduce potential for plugging of thefilter 468. As shown in FIGS. 12-15, one or both of the propellers 470may have pitched blades that direct flow of fluid from the respectivepropeller 470 in an axial direction relative to the axis of rotation ofthe rotatable shaft 472. As shown in FIGS. 14 and 15, the configurationof the bottom propeller 470 a may include impeller blades 494 that arepitched at an angle that will propel fluid flow in an upper axialdirection along the rotatable shaft 472 directed toward the tissuecollector 482 when the rotatable shaft 472 is rotated in the rotationaldirection 488. This type of upward pumping action by the propeller 470 amay assist in moving stringy tissue away from the filter 468 and towardthe tissue collector 482 to engage and collect on the members 486. Insimilar manner, as shown in FIGS. 14 and 15, the top propeller 470 b mayhave pitched blades 493 that propel fluid flow in an axial directionupward toward the underside of the lid 454 and away from the tissuecollector 482 when the rotatable shaft 472 is rotated in the rotationaldirection 488. This upward pumping action by the propeller 470 b mayassist in pulling tissue through the tissue collector 482 to promotecollection of stringy tissue by the members 486. As a designenhancement, the tissue collection members 486 may extend in an radialdirection outward from the axis of the rotatable shaft 472 to a greaterdistance than either one of or both of the blades of the propellers 470a,b. In particular, it is preferred that the members 486 may extend in aradial direction a distance that is beyond the radial distance of amaximum extent of the blades 494 of the bottom propeller 470 a, and in afurther enhancement the members 486 may extend in a radial directionfarther than a maximum extent of the blades 493, 494 of either of thepropellers 470 a and 470 b. In this way the members 486 may beconfigured to collect stringy tissue beyond the radial extent of one orboth of the propellers 470 a,b.

In one enhancement, one or more of the blades 494 may be configured toscrape at least a portion of the filter 468 when the rotatable shaft472, and thus also the bottom propeller 470 a, is rotated in therotational direction 488. In the configuration shown in FIGS. 12-15,such scraping of the filter 468 may be accomplished by configuring abottom edge portion 495 and/or slanted edge portion 496 of a blade 494to contact and scrape surfaces of the filter 468. In that regard, theslanted edge portion 496 of a blade 494 may be configured to correspondwith and contact a corresponding tapered portion of the filter 468. Aleading edge of the blade 494 may have a tapering width to assist inscraping tissue away from the surface of the filter 468. For example,the configuration of the blade 494 as shown in FIG. 14 includes abeveled surface 497 toward a leading edge of the slanted edge portion496 that may help to lift tissue away from the filter 468 when the lowerpropeller 470 a is rotated in the rotational direction 488.

As shown in FIGS. 14 and 15, the teeth 490 of the members 486 may havebeveled surfaces toward a leading edge that facilitate more easyrotation of the tissue collector 482 through tissue that may be disposedin the tissue retention volume of the container of the apparatus 450.

A rotatable tissue collector, for example as shown in FIGS. 14 and 15,may preferably be used in combination with at least one mixing impellerand/or in combination with at least two mixing impellers (as shown inexample of FIGS. 14 and 15). Alternatively, the tissue collector may beused by itself and not in combination with any separate mixing impeller.When a separate mixing impeller is used, one or more such separatemixing impeller may include one or more pitched blades (e.g., blades494, 493 of propellers 470 a,b of FIGS. 14 and 15) that impart axialflow for mixing, or one or more such separate mixing impeller mayinclude one or more unpitched blades that impart radial flow (e.g.,mixing members 320 of FIG. 4). When a rotatable tissue collector is usedalone, without any separate mixing impeller, the tissue collector byitself may serve also as a rotatable mixer with the tissue collectionmembers also acting as mixing members to mix contents within a tissueretention volume. In a preferred implementation, a rotatable tissuecollector is used in combination with at least one mixing impeller. Whenonly one mixing impeller is present, it is more preferred to dispose themixing impeller at a lower elevation on a rotatable shaft (e.g.,propeller 470 a of FIGS. 14 and 15) although an alternative arrangementis to include the mixing impeller at a higher elevation on a rotatableshaft (e.g., propeller 470 b of FIGS. 14 and 15). A preferredconfiguration is for each mixing impeller and tissue collector to becoaxial and connected to and rotatably driven by a single rotatableshaft, although such a single shaft arrangement is not required. Forinstance, one or more mixing impellers may be connected to and driven byone or more rotatable shafts separate from a rotatable shaft that drivesa rotatable tissue collector. A tissue collection and processingapparatus may have multiple rotatable tissue collectors, which may havethe same or different configurations and may be driven by the same ordifferent rotatable shafts.

The teeth and adjacent open spaces on tissue collection members of arotatable tissue collector may have a variety of configurations. It isnot necessary that the teeth be of the same height or configuration orthat the open spaces be of the same size or configuration, either on thesame tissue collection member or on different tissue collection members.

Reference is made to FIGS. 16 and 21-24 showing some exampleconfigurations for open spaces and teeth for a tissue collection member.Reference numerals corresponding with the tissue collection members,teeth and open spaces are the same as used in FIGS. 14 and 15, forconvenience of description. FIGS. 16 and 21-24 show five exampleconfigurations showing some examples for different configurations forteeth 490 and open spaces 492 for a tissue collection member 486.Examples of FIGS. 16, 21 and 22 all have teeth 490 that have a height h(distance between the top of a tooth 490 and the bottom of an adjacentopen space 492) that is equal for all teeth 490. The example of FIG. 23shows a configuration in which some teeth 490 have a greater height h₂than the height h₁ of some other teeth 490. The example of FIG. 24 showsa configuration with three different tooth heights (h₁, h₂, h₃).Examples of FIGS. 23 and 24 also show configurations in which not all ofthe teeth 490 and the open spaces 492 are configured with the samegeometry.

Reference is made to FIG. 17, which shows another embodiment for atissue collection and processing apparatus. FIG. 17 shows an apparatus600 including a lid 602 and a shell 604 that form a container having aninternal containment volume including a tissue retention volume 606 anda filtrate volume 608 disposed on different sides of a filter 610. Theapparatus 600 is shown in an access orientation as it would be supportedby base supports 611 that are integrally formed with the shell 604. Asuction port 612 is in fluid communication with the filtrate volume 608and through which material may be removed from the filtrate volume 608.An inlet port 614 is provided for introducing tissue or other materialinto the tissue retention volume 606. The apparatus 600 may includeadditional access ports parts (e.g., additional suction port, auxiliaryport), such as described for previous embodiments. The apparatus 600includes a rotatable assembly 616 including a mixing impeller 618 and atissue collector 620. The mixing impeller 618, tissue collector 620 anda spacer 622 are mounted on a rotatable shaft (not shown) that extendsfrom above the container through the lid 602 and into the tissueretention volume 606. The rotatable shaft includes a central lumen thatextends through the rotatable shaft from outside the container and opensat the bottom of the mixing assembly near the bottom portion of thetissue retention volume 606 just above the filter 610. The lumen isaccessible by removing a cap 624. The lid 602, shell 604, tissueretention volume 606, filtrate volume 608, filter 610, base supports611, suction port 612, inlet port 614, rotatable shaft, mixing impeller618 and tissue collector 620 may have any design features orconfigurations as described previously in relation to correspondingfeatures of the apparatus described in any of FIGS. 1-16 and 21-24. Theapparatus 600 does, however, include a pellet well 626 at the bottom ofthe filtrate volume 608, as discussed further below.

In some preferred implementations, the tissue collector 620 may have adesign similar to the corresponding tissue collector described withrespect to FIGS. 14-16 and 21-24 and the mixing impeller 618 may beconfigured with pitched blades for producing axially upward flow towardthe tissue collector 620 when the rotatable shaft is rotated in anappropriate direction. The blades of the mixing impeller 618 maybeneficially be designed with portions that scrape the filter 610 as therotatable shaft 616 is rotated, in a manner similar as described abovewith respect to FIGS. 14-16 and 21-24. The lumen through the rotatingshaft may be aligned with a collection volume located in the filtratevolume 608 below the bottom of the filter 610, and may provide accessfor convenient removal of processed material from the pellet well 626 inthe collection volume located below the filter 610.

Various example dimensions are shown for the apparatus 600. A firstheight dimension D₁ shows the vertical dimension from the bottom of thecollection volume at a nadir of the filtrate volume 608 to a topelevation of the collection volume occupied by the pellet well 626.Second height dimension D₂ shows the vertical dimension from the bottomto the top of the collection volume that is below the filter 610. Thirdheight dimension D₃ shows the vertical dimension from the bottom of thefilter 610 to the bottom of a skirt 628 from which the filter 610 issuspended. Fourth height dimension D₄ shows the vertical extent of theskirt 628. Angle A₁ is an angle between horizontal and a first taperedinterior wall surface 630 of the container that defines at least aportion of the filtrate volume 608, including defining at least aportion of the collection volume. Angle Az is an angle from horizontalto a second tapered interior wall surface 632 of the container thatdefines at least a portion of the pellet well. Angle A₃ is an anglebetween horizontal and a third tapered interior wall surface of thecontainer that defines at least a bottom portion of the pellet well 626.Example dimensions for one example implementation for the embodiment ofthe apparatus 600 includes 25.7 millimeters for D₁, 37.1 millimeters forD₂, 55.9 millimeters for D₃, 7.9 millimeters D₄, 45° for A₁, 80° for A₂,and 30° for A₃. Such an example may be designed for example to includean internal containment volume of about 270 cubic centimeters and avolume in the pellet well 626 of about 1.2 cubic centimeters, and withthe tissue retention volume 606 configured to accommodate processing ofabout 110 cubic centimeters of adipose tissue in the tissue retentionvolume 606 for preparation of a pellet phase including leuko stromalvascular fraction concentrate that may fill or nearly fill the pelletwell 626.

The location and configuration of the pellet well 626 in the embodimentof the apparatus 600 shown in FIG. 17 facilitates direct aspiration ofmaterial of a pellet phase that may collect in the pellet well 626 byproviding a relatively deep and narrow chamber that helps facilitateeffective aspiration of the pellet phase material without alsoaspirating large quantities of overlying material from less-densematerial phases that may form during centrifuge processing.

FIG. 18 shows the apparatus 600 of FIG. 17 in which a hypodermic needle640 is inserted through the lumen of the rotatable shaft to accesspellet phase material 642 from above for direct aspiration of the pelletphase material 642 from the pellet well 626 through the hypodermicneedle 640 to outside of the container and into a syringe 644. Suchdirect aspiration of the pellet phase material 642 may be performedwithout first removing less-dense material 646 from above the pelletphase material 642 and without suspension of the pellet phase material646 in a suspension liquid. The syringe 644 may be preloaded with aquantity of dispersion medium that mixes with and disperses aspiratedpellet phase material as it is introduced into the syringe 644. This mayhelp prevent clumping of the pellet phase material in the syringe. Theresulting mixture of pellet phase material and dispersion medium may beremoved and further processed to prepare a composition foradministration to a patient or the mixture may be directly administeredto a patient as a delivery composition, such by injection into a patientin the vicinity of a joint to treat for osteoarthritis at the joint. Ifa mixture in the syringe is removed from the syringe 644 for furtherprocessing, the mixture may be centrifuged to separate pellet phasematerial and suspension liquid and the separated pellet phase materialmay be recovered and formulated with other components in a deliverycomposition, which may for example include a scaffold material or mayinclude dispersion in a new dispersion medium with properties and at avolume desired for a particular treatment application. Any of the wallsurfaces defining at least a portion of any of the first taperedportion, second tapered portion and third tapered portion may haveinclined planar geometry with a constant angle of inclination, as shownin FIGS. 17 and 18 for angles A₁, A₂ and A₃ or may have a curvedgeometry with a changing angle of inclination. When such a surface has acurved geometry, the respective angle, may be, may be the angle ofinclination of a line tangent to a point on the curved geometry.

FIG. 19 is a generalized process block diagram illustrating oneembodiment of a method involving multi-step processing within a portablecontainer, such as for example using a tissue collection and processingapparatus as previously described. As shown in FIG. 19, the methodincludes a washing step 502, during which adipose tissue disposed withina portable container is washed to remove contaminants from the adiposetissue. Contaminants that may be associated with the adipose tissueinclude for example blood, free lipids, small particles and debris andother materials that may have been collected with the adipose tissue orresult from degradation during a tissue collection operations.

The washing 502 may include one or multiple wash cycles during whichadipose tissue is washed with wash liquid within the container. The washliquid, for example, may be a buffer solution, such as Lactated Ringer'ssolution or Hank's Balanced Solution, and may have additional additives,such as one or more of an anti-clotting agent, an antibiotic and anantifungal. An anti-clotting agent may beneficially prevent coagulationof blood that may be present, and may assist effective washing of bloodfrom the adipose tissue. Antibiotics and antifungals may help protectagainst problems associated with inadvertent outside contamination ofthe adipose tissue within the container. Such a wash liquid may alsoinclude one or more additional buffering agents, such as glycine. Onepreferred material for use as an anti-clotting agent is heparin.

During a wash cycle, the wash liquid is mixed with the adipose tissue inthe container and then preferably substantially all of the wash liquidwith washed contaminants from the adipose tissue is removed from thecontainer from a filtrate volume on a first side of a filter within thecontainer while retaining the washed adipose tissue in a tissueretention volume of the container on a second side of the filter.

The washing may include any of the features discussed above.

After the washing 502, the washed adipose tissue in the container issubjected to a digesting step 504. Digestion medium, such as comprisinga collagenase enzyme solution, is added to the container to contact thewashed adipose tissue. The digestion medium may for example be added ina volume ratio of in a range of from 0.6:1 to 2:1 digestionmedium:adipose tissue. The digestion medium may contain collagenaseenzyme, for example in an amount to provide from 150 to 300 collagendigestion units (CDU) per milliliter of catalytic volume. Catalyticvolume refers to the total volume of the digestion medium and adiposetissue within the container to which the digestion medium is added.After the digestion medium is added to the container, enzymaticdigestion within the container is permitted to proceed for a retentiontime, for example, of from 20 minutes to 50 minutes while the containeris disposed in a temperature controlled environment maintained within atemperature range preferably of from 32° C. to 38° C., and with at leastoccasional, and preferably substantially continuous, agitation ofcontents to the container. The digesting step 504 may include any or anycombination of the feature refinements and additional features discussedabove.

The method as shown in FIG. 19 also includes a stopping digestion step506 occurring after the digesting step 504. The stopping digestion step506 should preferably occur no earlier than the end of the retentiontime for the enzymatic digestion in the temperature controlledenvironment, but in any event should more preferably be performed within50 minutes following adding the digestion medium to the container duringthe digesting step 504. The stopping digestion step 506 includes addinga stopping reagent to the container to positively stop enzymaticactivity within the container. This is important, because if enzymaticactivity is not discontinued, digestion within the container may proceedto an undesirable degree in which the enzyme may destroy the viabilityof a significant number of the leuko stromal vascular cells.

As shown in FIG. 19, the method includes, after the stopping digestionstep 506, a centrifuging step 508. The centrifuging step 508 isperformed with the container disposed in a centrifuge and the centrifugeis operated to centrifuge the container to form density-separated phaseswithin the container. These density-separated phases include ahigher-density pellet phase rich in leuko stromal vascular cells, whichpellet phase may form adjacent a bottom of the container. Thedensity-separated phases also include lower-density material phases. Bylower-density, it is meant that the lower-density material phases have alower-density than the pellet phase. When the container is oriented withthe pellet adjacent a bottom of the container (e.g., in an accessorientation for the container), the lower-density material phases willbe disposed in the container above the pellet phase. The lower-densitymaterial phases may include, in order of decreasing density, an aqueouslayer, a disaggregated adipose layer (containing remnants ofdisaggregated adipose tissue) and an oil layer. The pellet phase isenriched in, and may be mostly or even substantially entirely comprisedof, leuko stromal vascular cells (e.g., stromal vascular fraction). On aside of the pellet phase opposite the lower-density material phases maybe disposed a small red blood cell phase. Provided that washing of theadipose tissue is thorough during the washing step 502, this red bloodcell phase may be extremely small, and in some case may be difficult todistinguish from a bottom portion of the pellet phase. The centrifugingstep 508 may include any or any combination of the feature refinementsand additional features discussed above.

As shown in FIG. 19, the stopping digestion step 506 is performed afterthe digesting step 504 and prior to the centrifuging step 508. Suchsequencing is preferred, but not required. In one variation, thestopping digestion step 506 may be performed after the centrifuging 508.However, because enzymatic digestion would continue during thecentrifuging, such a variation in the sequence is not preferred, toprovide better control over the timing and extent of the enzymaticdigestion.

After the centrifuging step 508 has been completed, the container may beremoved from the centrifuge and subjected to a step 510 of selectivelyremoving pellet phase material. The leuko stromal vascular cells, whichinclude stem cells, contained in the pellet phase represent a valuableproduct. For effective use of these valuable leuko stromal vascularcells, it is generally necessary to remove the cells from the container.This has been a significant problem in the context of using multi-stepportable containers for processing that is addressable with variousimplementations of the invention. During the step 510, material of thepellet phase is removed from the internal containment volume of thecontainer to outside of the container separate from the less-densematerial phases. The step 510 may include any of the features asdiscussed above. In some processing alternatives, the pellet phasematerial may be directly aspirated through an aspiration tube (e.g.,hypodermic needle) inserted into the pellet phase from above andmaterial of the pellet phase may be directly aspirated from thecontainer through the aspiration tube, for example into a syringe orother fluid receptacle located outside of the container.

Referring now to FIG. 20, another embodiment of implementation of amethod is shown, including an alternative approach for selectivelyremoving material of the pellet phase from the container that. Theimplementation shown in FIG. 20 includes the washing step 502, thedigesting step 504, the stopping digestion step 506, the centrifugingstep 508 and the selectively removing pellet phase material step 510 asdiscussed with FIG. 19, but showing more detail for some processingalternatives for step 510. As shown in FIG. 20, the step 510 includessteps 511, 512 and 514. During the step 511, the lower-density materialphases formed during centrifuging may be removed from the containerwhile the pellet phase is retained within the container, preferablywhile maintaining the pellet phase in an undisturbed state, in place atthe location of the container where the pellet collected during thecentrifuging. The step 511 may include any or any combination of thefeatures as discussed above. As shown in FIG. 20, after the removinglower-density phases step 511, the method includes a dispersing cellsstep 512. During the dispersing cells step 512, aqueous suspensionliquid is introduced into the container to mix with the pellet phase andto act as a dispersion medium for dispersing cells of the pellet phasein the suspension liquid. Dispersion of cells from the pellet phase maybe aided by tapping the container to dislodge and break up the pelletphase to assist effective dispersion of the leuko stromal vascular cellsin the suspension liquid. The dispersing cells step 512 may include anyof the features as discussed above. After the dispersing cells step 512,the processing shown in FIG. 20 includes a removing dispersed cells step514, during which most, and preferably substantially all, of thesuspension liquid with the dispersed cells from the pellet phase isremoved from the container, thereby recovering the leuko stromalvascular cell from the container. The removing dispersed cells step 514may include any of the features discussed above.

As an alternative to the processing for the selectively removing pelletphase material step 510 shown in FIG. 20, after the removinglower-density material phases step 511, the material of the pellet phasecould be removed from the container by direct aspiration through anaspiration tube, such as a hypodermic needle. With this processingalternative, the cells of the pellet phase material would not bedispersed or suspended in a suspension liquid prior to removal from thecontainer.

The foregoing discussion of the invention and different aspects thereofhas been presented for purposes of illustration and description. Theforegoing is not intended to limit the invention to only the form orforms specifically disclosed herein. Consequently, variations andmodifications commensurate with the above teachings, and the skill orknowledge of the relevant art, are within the scope of the presentinvention. The embodiments described hereinabove are further intended toexplain best modes known for practicing the invention and to enableothers skilled in the art to utilize the invention in such, or other,embodiments and with various modifications required by the particularapplications or uses of the present invention. It is intended that theappended claims be construed to include alternative embodiments to theextent permitted by the prior art. Although the description of theinvention has included description of one or more possibleimplementations and certain variations and modifications, othervariations and modifications are within the scope of the invention,e.g., as may be within the skill and knowledge of those in the art afterunderstanding the present disclosure. It is intended to obtain rightswhich include alternative embodiments to the extent permitted, includingalternate, interchangeable and/or equivalent structures, functions,ranges or steps to those claimed, whether or not such alternate,interchangeable and/or equivalent structures, functions, ranges or stepsare disclosed herein, and without intending to publicly dedicate anypatentable subject matter. Furthermore, any feature described or claimedwith respect to any disclosed implementation may be combined in anycombination with one or more of any other features of any otherimplementation or implementations, to the extent that the features arenot necessarily technically compatible, and all such combinations arewithin the scope of the present invention.

The terms “comprising”, “containing”, “including” and “having”, andgrammatical variations of those terms, are intended to be inclusive andnonlimiting in that the use of such terms indicates the presence of somecondition or feature, but not to the exclusion of the presence also ofany other condition or feature. The use of the terms “comprising”,“containing”, “including” and “having”, and grammatical variations ofthose terms in referring to the presence of one or more components,subcomponents or materials, also include and is intended to disclose themore specific embodiments in which the term “comprising”, “containing”,“including” or “having” (or the variation of such term) as the case maybe, is replaced by any of the narrower terms “consisting essentially of”or “consisting of” or “consisting of only” (or the appropriategrammatical variation of such narrower terms). For example, the astatement that some thing “comprises” a stated element or elements isalso intended to include and disclose the more specific narrowerembodiments of the thing “consisting essentially of” the stated elementor elements, and the thing “consisting of” the stated element orelements. Examples of various features have been provided for purposesof illustration, and the terms “example”, “for example” and the likeindicate illustrative examples that are not limiting and are not to beconstrued or interpreted as limiting a feature or features to anyparticular example. The term “at least” followed by a number (e.g., “atleast one”) means that number or more than that number. The term at “atleast a portion” means all or a portion that is less than all. The term“at least a part” means all or a part that is less than all.

What is claimed is:
 1. A method for preparing a treatment composition for treatment of osteoarthritis including un-cultured stromal vascular fraction cells from adipose tissue, the method comprising: direct aspiration of material of a centrifugally-formed pellet phase from an internal containment volume of a container of a portable apparatus, the material of the pellet phase including stromal vascular fraction cells from enzymatically-digested adipose tissue, the direct aspiration comprising aspiration of at least a majority of the material of the pellet phase through an aspiration tube inserted into the pellet phase and into a fluid receptacle in fluid communication with the aspiration tube and located outside of the container.
 2. A method according to claim 1, wherein the fluid receptacle is pre-loaded prior to the direct aspiration with dispersion medium that mixes with the pellet phase material introduced into the fluid receptacle during the direct aspiration.
 3. A method according to claim 2, wherein the dispersion medium pre-loaded in the fluid receptacle has a volume in a range of from 1 milliliter to 10 milliliters.
 4. A method according to claim 3, wherein a volume ratio of the volume of the dispersion medium pre-loaded in the fluid receptacle to a volume of the pellet phase material introduced into the fluid receptacle during the direct aspiration is in a range of from 1:1 to 10:1.
 5. A method according to claim 2, wherein the dispersion medium comprises one or more than one member selected from the group consisting of saline solution, crystalloid solution, hyaluronic acid and hyaluronic acid-based materials.
 6. A method according to claim 1, wherein: during the direct aspiration, the portable apparatus is oriented in an access orientation; the internal containment volume includes a pellet well located in a bottom portion of the internal containment volume and accessible only from above when the portable apparatus is in the access orientation; during the direct aspiration, the at least a majority of the pellet phase material is aspirated from the pellet phase disposed in the pellet well with the aspiration tube inserted from above the pellet well into the pellet phase in the pellet well.
 7. A method according to claim 6, wherein during the direct aspiration, the aspiration tube is inserted into the internal containment volume from above the container and the aspiration tube extends downward through the internal containment volume into the pellet well.
 8. A method according to claim 7, wherein: the pellet well has a volume in a range of from 0.3 cubic centimeter to 3 cubic centimeters; when the portable apparatus is in the access orientation, the pellet well has a vertical height dimension in a range of from 1 centimeter to 10 centimeters; when the portable apparatus is in the access orientation, the pellet well has at least one portion with a vertical length of 1 centimeter, a maximum horizontal dimension along the vertical length of no larger than 5 millimeters and a minimum horizontal dimension along the vertical length of no smaller than 1.5 millimeters; and during the direct aspiration, the aspiration tube extends into a said portion of the pellet well.
 9. A method according to claim 8, wherein: the portable apparatus comprises a filter disposed within the internal containment volume and wherein the internal containment volume includes a tissue retention volume on one side of the filter and a filtrate volume on another side of the filter with the tissue retention volume and the filtrate volume being in fluid communication through the filter and with the pellet well disposed in a bottom portion of the filtrate volume; during the direct aspiration the aspiration tube passes through the tissue retention volume and the filtrate volume and pierces the filter.
 10. A method according to claim 9, wherein; the internal containment volume includes a first tapered portion below a bottom elevation of the filter and above the pellet well when the portable apparatus is in the access orientation, the first tapered portion being defined at least in part by a first internal wall surface at a first angle relative to horizontal when the apparatus is in the access orientation in a range of from 20° to 65°; the internal containment volume includes a second tapered portion in the pellet well below the first tapered portion, wherein the second tapered portion is defined at least in part by a second internal wall surface having a second angle relative to horizontal when the apparatus is in the access orientation in a range of from 70° to 89°; the internal containment volume includes an available processing volume with a portion of the available processing volume disposed in the tissue retention volume and another portion of the available processing volume in the filtrate volume; and the second tapered portion of the internal containment volume in the pellet well has a volume in a range of from 0.2 percent to 2.5 percent of the portion of the available processing volume in the tissue retention volume.
 11. A method according to claim 10, wherein the available processing volume is in a range of from 75 cubic centimeters to 1300 cubic centimeters.
 12. A method according to claim 9, wherein the portable apparatus comprises a tissue collector disposed in the tissue retention volume and rotatable relative to the container in at least a first direction of rotation about an axis of rotation of a rotatable shaft, the tissue collector including at least one toothed member that sweeps through a portion of the tissue retention volume when the tissue collector is rotated in the first direction, the toothed member being configured with a plurality of teeth to collect and retain stringy tissue when the tissue collector is rotated in the first direction in contact with human biological material containing stringy tissue disposed in the tissue retention volume.
 13. A method according to claim 1, comprising prior to the direct aspiration: washing adipose tissue within the container to remove contaminants from the adipose tissue; after the washing, digesting adipose tissue within the container, the digesting adipose tissue comprising adding to the container a volume of enzyme-containing digestion medium to contact washed adipose tissue in the container; after permitting enzymatic digestion in the container for a retention time following adding the digestion medium, disposing the container in a centrifuge and centrifuging the container in the centrifuge to form density-separated phases within the container, the density-separated phases including the pellet phase; and inserting the aspiration tube from outside of to inside of the container to contact the pellet phase in the internal containment volume within the container.
 14. A method for treating a patient for osteoarthritis of a joint, the method comprising: preparing a treatment composition according to the method of claim 1, wherein the treatment composition comprises material of the pellet phase dispersed in a dispersion medium; administering into the joint or to a vicinity of the joint an administration volume of the treatment composition, wherein the administration volume is in a range of from 0.5 milliliter to 5 milliliters and includes from 0.25 millimeter to 2.5 milliliters of the pellet phase material dispersed in the dispersion medium.
 15. A method according to claim 14, wherein the administering comprises injecting the treatment composition into the joint or to the vicinity of the joint from the fluid receptacle into which the pellet phase material is introduced during the direct aspiration.
 16. A method according to claim 15, wherein the dispersion medium of the treatment composition comprises dispersion medium pre-loaded into the fluid receptacle prior to the direct aspiration.
 17. A method according to claim 15, wherein the dispersion medium comprises one or more than one member selected from the group consisting of saline solution, crystalloid solution, hyaluronic acid and hyaluronic acid-based materials.
 18. A method according to claim 14, wherein: during the direct aspiration, the portable apparatus is oriented in an access orientation; the internal containment volume includes a pellet well located in a bottom portion of the internal containment volume and accessible only from above when the portable apparatus is in the access orientation; and during the direct aspiration, the at least a majority of the pellet phase material is aspirated from the pellet phase disposed in the pellet well with the aspiration tube inserted from above the pellet well into the pellet phase in the pellet well.
 19. A method according to claim 18, wherein the portable apparatus comprises a filter disposed within the internal containment volume and wherein the internal containment volume includes a tissue retention volume on one side of the filter and a filtrate volume on another side of the filter with the tissue retention volume and the filtrate volume being in fluid communication through the filter and with the pellet well disposed in a bottom portion of the filtrate volume; the internal containment volume includes a first tapered portion below a bottom elevation of the filter and above the pellet well when the portable apparatus is in the access orientation, the first tapered portion being defined at least in part by a first internal wall surface at a first angle relative to horizontal when the apparatus is in the access orientation in a range of from 20° to 65°; the internal containment volume includes a second tapered portion in the pellet well below the first tapered portion, wherein the second tapered portion is defined at least in part by a second internal wall surface having a second angle relative to horizontal when the apparatus is in the access orientation in a range of from 70° to 89°; the internal containment volume includes an available processing volume with a portion of the available processing volume disposed in the tissue retention volume and another portion of the available processing volume in the filtrate volume; and the second tapered portion of the internal containment volume in the pellet well has a volume in a range of from 0.2 percent to 2.5 percent of the portion of the available processing volume in the tissue retention volume.
 20. A method according to claim 19, wherein during the direct aspiration the aspiration tube is inserted into the internal containment volume from above and passes through the tissue retention volume and the filtrate volume and pierces the filter. 